Article (6) - 2009 - Arien-zakay Et Al

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 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 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: [email protected] 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 differentiation 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,

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such as growth, branching, differentiation, and synapse formation were found to be influenced by different matrixresident biophysical cues such as micro- and nanotopography (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 neuronalcontaining scaffolds and histological evaluation of the morphology of the fixed cells, allowing temporal “snapshots” 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 morphological 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.

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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) supplemented 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 threeplasmid 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.

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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.

clone and wild-type cells. GFP expression was estimated by fluorescence microscopy. Clone adherence was assessed by monolayer generation; nonadherent cells grew in suspension 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.

FACS Sorting

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.

Flow cytometric analysis of the PC12-GFP/lentivirustransduced 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 (BectonDickinson). 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

2-D Cell Growth Conditions

3-D Cell Growth Conditions Sandwiched 3-D type I collagen gels at a final concentration 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,

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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 collagencoated 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

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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 leastsquares 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 manufacturer’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: denaturation of cyclic parameters at 94°C for 1 min, annealing at 65°C for 1 min, and elongation at 72°C for 2 min (ArienZakay 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

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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 phosphorylated ERK was performed overnight at 4°C using rabbit monoclonal anti-phospho-p44/42 MAPK (extracellular signalregulated 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/horseradishperoxidase-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 nonphosphorylated protein, using a two-step procedure similar to that previously described (Takman et al. 2004). Quantitative analysis of ERK phosphorylation activity was performed 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 microscope (Olympus 300 IX-70, Japan and Axiovert 200, Zeiss, Göttingen, Germany), using excitation and emission wavelengths 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 experimental groups were determined by analysis of variance; the results were considered significant when p<0.05.

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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 GFPtransduced 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 transfected 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 proliferation 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-

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Figure 1 Development and characterization of stable GFPPC12 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 wtPC12 (white) under 2-D conditions, 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, resulting in neuronal differentiation as evidenced by neurite outgrowth (Kaplan and Miller 2000). To quantitatively evaluate the NGF-induced TrkA activation in the GFPPC12 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

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Table 1 Biological characterization of four different GFP-PC12 clones PC12 clonea

Proliferation (doubling time, h)b

GFP expression (fluorescence intensity)c

Morphologyd

Adherencee

NGF response (% of cells with outgrowths)f

wt-PC12 PC12-4c PC12-1d PC12-4d PC12-4f

48 50–60 50–60 70–80 50–60

0 103 105–106 104 105

Neuronal Neuronal Neuronal Heterogeneousg Neuronal

Monolayer Monolayer Monolayer Monolayer Monolayer

100 80 60 100 100

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 untreated as control. In parallel experiments, wt-PC12 cells at the same density were also treated under identical conditions. Upon NGF treatment, the majority of the

GFP-PC12 cells extended neurite outgrowths of different lengths (Fig. 3a, lower panel) similar to wt-PC12 cells. A typical dose response of the NGF-induced differentiation is presented in Fig. 3b. The similarity of the response in GFP-

Figure 2 Characterization of NGF receptor mRNA transcript expression 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 phosphorylated (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

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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 dosedependent differentiation response, measured by elongation of neurite outgrowths (E), as described in “Materials and Methods”; gray bars

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

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 quantitatively that NGF-induced differentiation of GFP-PC12 is very similar to that of the wt-PC12 cells. Therefore, GFPPC12 cells provide a new valid model for the visualization and imaging of neurite extension as a surrogate marker of neuronal differentiation.

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 aggregation, 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

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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

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 outgrowths 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, outgrowths 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 elongation 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

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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-

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 dosedependent differentiation responses measured using Df and E. The values represent the mean ± SEM of quadruplicate experiments. *p< 0.01 vs untreated cells

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 NGFinduced differentiation of GFP-PC12 cells in 3-D beyond that observed at day 15.

Discussion

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

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 GFPtransgenic 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

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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 GFPPC12 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 overexpressing the tissue plasminogen activator, which promotes 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 conventional 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

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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 observations 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 quantitative 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 neurotrophin-induced differentiation in/on 3-D polymeric scaffolds (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 conjunction with near-infrared multiphoton microscopy and in vivo laser tomography, should provide novel models for drug screening and high-resolution structural imaging (SchenkeLayland 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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