ANAT3212 Research Project 2012

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Introduction

There are many neuronal cell lines available for the study of cell differentiation under differing conditions. The cell types that were used in this study were types of neuronal cells derived from the retina, neuroblastomas and pheochromocytomas.

Cell Types

The first type of cells used in this study was RGC-5 cells. These cells are retinal ganglion cells which were originally thought to be a rat retinal ganglion cell line. In 2009, however, an article was published with research using mitochondrial and nuclear DNA analyses, showing that RGC-5 cells were not in fact rat cells but of mouse origin [1]. Morphologically, the undifferentiated cells look epithelial and have short extensions.


The B35 cell line is derived from neuroblastoma induced by nitrosoethylurea (NEU) in newborn rats. Tumours that were found in the central nervous system of newborn rats at age 4-10months after birth were excised, minced, adapted to culture and then cloned[2]. The morphology of these undifferentiated cells is that they are small round neurons, with extending neurites that are sometimes branched. The cells may also be bipolar in appearance.


The final cell type used in this study was PC12 which is derived from a pheochromocytoma of the rat adrenal medulla. The cells are small (5-10 µm), have a limited amount of cytoplasm and have a long doubling time of approximately 2 days or more (Kelly-Spratt, 1998). In terms of morphology, the undifferentiated cells are small and irregularly shaped. These cells tend to grow in floating clusters typically with a minority of the cells being scattered and lightly attached.

Growth Factors

Brain Derived Neurotrophic Factor (BDNF) is a secreted protein that is encoded by the BDF gene in humans. It is a member of the neurotrophin family of growth factors and acts on neurons in the Central Nervous System (CNS) and Peripheral Nervous system (PNS) to help support the survival of existing neurons and encourage growth and differentiation of new neurons and synapses [3]. It has also been found to be active in the hippocampus, cortex, and the basal forebrain which are all important areas related to memory and complex thinking [4]. It is known that BDNF binds to receptors on the surface of cells capable of responding to the growth factor. The two known receptors are TrkB and low-affinity nerve growth factor (LNGFR) [5].


Nerve Growth Factor (NGF) is a small secreted protein that is involved in the growth and maintenance of nerve cells particularly of sympathetic and sensory neurons. It is also involved in the repair, regeneration and protection of neurons. Therefore, it could be used as a therapeutic agent in the treatment of different neurodegenerative diseases such as Alzheimer’s disease. NGF binds with at least 2 classes of receptors including TrkA which is a high-affinity tyrosine kinase receptor[6]. This phosphorylates the TrkA which activates numerous other signalling pathways leading to a change of gene expression. BDNF and NGF both work on surface receptors while retinoic acid works on intracellular receptors.


Retinoic Acid acts by binding to the retinoic acid receptor (RAR) which is bound to DNA with the retinoid C receptor (RXR) in regions called retinoic response elements (RAREs). It is a metabolite of vitamin A and controls the functions of vitamin A which are involved in regular growth and development (Holland, 2007). It is particularly important during embryonic development where it is involved in patterning in the early developmental stages through Hox genes [7].


Staurosporine is a potent and cell permeable alkaline which is isolated from the culture of Streptomyces staurosporesa. It is an inhibitor of protein kinase C at a concentration of 0.7nm and at higher concentrations of approximately 1-20nm it is also an inhibitor of PKA, PKG, CAMKII & MLCK which are other kinases [8]. Couldwell et al (1994) found that at approximately 50 – 100nM, Staurosporine is a neurotrophin agonist and promites neurite growth in neuroblastoma, brain primary neuronal cultures and pheochromocytoma [9]. It has also been found to induce cell apoptosis at 0.2-1 μM [10].

Aim and Hypothesis

This study aimed to investigate the effects these different growth factors BDNF, NGF, Retinoic Acid and Staurosporine had on the differentiation of RGC-5, B35 and PC-12 cells. Another aim of this study was to investigate working in a laboratory of an active research group and to experience the various methods and procedures used when working in a cell biology lab. The null hypothesis assumes that the neuronal growth factors will have no effect on the differentiation of these cells in terms of the cell counts, the morphology of the cell and the number of differentiated cells with processes.

Method

Each cell line was stored according to their passage numbers in liquid nitrogen. The cells were retrieved from here and double contained to be transported back to the lab in order to be transferred to the culture dishes. Each cell type then needed to be thawed in a water bath before being transferred to the culture dishes which contained 8ml of medium. The medium was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with high glucose and Penicillin-Streptomycin Solution. The cells were than incubated at 37 with 5% CO2 overnight. The cells were then able to be imaged over the next two days in order to investigate the characteristics of the undifferentiated cells.


After two days the cells had proliferated sufficiently for the cells to be transferred from the culture dishes to 24 well plates where the different growth factors were to be added later. In order to remove the cells from the culture dishes the medium had to be removed using a vacuum pump and Ca/Mg free PBS was then added to remove the media growth factors. The next step was adding tripsone which is a proteaze that breaks up proteins and removes calcium. This meant the cell adhesions were able to be broken allowing the cells to be transferred to the wells. The cells were only able to be exposed to the Tripsone for 1 min at 37degrees as if it was left any longer the tripsone would continue to break up the proteins and destroy the cells. A small amount of serum was added after the minute passed so that the Tripsone could digest these proteins instead the proteins within the cells.


The bottom of the cell culture dishes was manually tapped in order to shift the cells from the bottom of the dish. They were then transferred into tubes before being placed in a centrifuge for 5 minutes to settle the cells at the bottom in a pellet. The liquid on top of the pellet was then aspirated off and the cells were resuspended in approximately 5m of medium. The pellet was dislodged by carefully drawing the liquid up and down using a disposable pipette. Once the total number of cells in a restricted volume area had been counted using a hemocytometer it was possible to calculate the amounts that needed to be added to each well so that the total volume in each well would be approximately 300ul.


The 24 well plates were then put into the incubator and the next day the medium was changed and the four growth factors were added. BDNF, NGF, Retinoic Acid and Staurosporine were added to a total of 4 wells for each of the three cell types (RGC-5, B35 and PC-12). Each cell type also had 4 wells of control where no new growth factor was added and 4 wells of a control that was plated at half the cell density. On the 3rd and 5th day the cells were observed using an inverted phase contrast microscope, counted and imaged.

Results

Average number of B35, PC12 and RGC5 cells per field of view at 4 days under Brain Derived Neurotrophic Factor, Nerve Growth Factor, Retinoic Acid and Staurosporine conditions.
4 days after the addition of growth factor to the cell types the control conditions had become confluent. The retinoic acid condition had a smaller cell count than the other non-control conditions with a cell count of 0 for the B35 cells.
Average number of B35, PC12 and RGC5 cells per field cof view at 4 days under Brain Derived Neurotrophic Factor, Nerve Growth Factor, Retinoic Acid and Staurosporine conditions.


The condition with the highest percentage of process growth was the staurosporine condition with more than 50% of cells with processes in all 3 cell types. 28.8% of B35, 37.3% of PC12 and 31.4% of RGC5 cells in the BDNF condition had grown processes. 43.8% of B35, 42.2% of PC12 and 67.4% of RGC5 cells in the NGF condition had grown processes. In the retinoic acid condition none of the B35 cells had grown processes. 26.3% of PC12 and 17.8% of RGC5 cells had grown processes. 57.0% of B35, 70.37% of PC12 and 71.9% of RGC5 cells had grown processes. In the control condition 7.2% of B35, none of the PC12 and 16.1% of cells had grown processes of some sort.


Average number of B35, PC12 and RGC5 cells per field cof view at 4 days under Brain Derived Neurotrophic Factor, Nerve Growth Factor, Retinoic Acid and Staurosporine conditions.
Average number of B35, PC12 and RGC5 cells per field cof view at 4 days under Brain Derived Neurotrophic Factor, Nerve Growth Factor, Retinoic Acid and Staurosporine conditions.
At 6 days the B35 cell count was at 0 for all growth medium conditions including the control condition. RGC5 cells had reduced by 0.5-0.75%. The PC12 cells had not survived 5 days in the growth factor and the cell count was 0 for the non-control groups. The control conditions for RGC5 and PC12 were confluent.


In the undifferentiated state, the B35 cell were triangular with few processes. The PC12 cells form clusters of spherical cells when undifferentiated and RGC5 cells are elongated and occasionally with processes. When differentiated, cells grow several longer processes with a triangular shaped neural cone at the end of the process. Cells are less abundant in the differentiated state and are therefore more easily identified for imaging compared to the more confluent state when cells are undifferentiated.

Discussion

After 3 days of incubation in the different growth factors the cells had shown to disprove the null hypothesis as the cells had stopped proliferation and a large amount of cells had started to differentiate. In the BDNF condition, there had been some differentiation but the prevention of proliferation was evident in the B35 and RGC5 cells. Cells in the NGF condition had survived and this condition had the largest average amount of cells counted per field of view as shown in table 1. Retinoic acid appears to have been at a concentration too high for the cells as the cells counts were lower than the other growth factor conditions. Staurosporine proved to be the most interesting factor due to the low cell count but high percentage of differentiated cells. For the purpose of this study any double layer extension of the membrane was counted as a process in the control condition and any extensions which contained a neuronal cone is counted as the process of a differentiated cell in the growth factor conditions.


The morphological changes which take place when the cells differentiate can be seen in figure 2 where the all cell types undergo similar changes such as the elongation of extensions into long processes. The three cell types grow characteristically different as they are different cell types. RGC5 cells were more circular and have one long process and several shorter processes. B35 cells appeared more triangular and often had longer processes that occasionally split again. PC12 cells were more spider-like and had several processes which extend from a more circular soma.


BDNF was shown in table 1 to have no effect on PC12 cells as they proliferated over the 4 days and 3 of the 4 wells had grown close to confluence. This was unexpected as PC12 cells are derived from the medulla which is part of the CNS. Since BDNF participates in supporting growth and survival of CNS and PNS neural cells it can only be concluded that the concentration of the BDNF was not high enough to activate and support the differentiation of all the PC12 cells hence, most of the cells continued to proliferate.


All cells in the NFG condition had shown healthy growth and by 4 days the majority had begun differentiation. The RGC5 cells had shown a high percentage of cell differentiation and this could be due to the NGF differentiation properties on sensory neurons in particular. As RGC5 cells are sensory then NGF should support the growth and maintenance of the RGC5 cells. B35 cells which are CNS cells had been supported by the NGF as B35 had the highest cell count in the NGF condition.


Retinoic acid had possibly been at a too high concentration as the B35 wells had shown very low amounts of cells which had survived. The PC12 cells had shown a high percentage of differentiated cells and this can be attributed to role retinoic acid plays in the patterns of development during the embryonic period. The importance of retinoic acid to vision can be seen in the survival of the RGC5 cells. The RGC5 cells had the highest cell count, they also happen to be cells involved in vision which means survival of cells would be supported by the retinoic acid which is known to act on vision during embryonic development.


The concentration of staurosporine in this experiment had been low enough to allow the survival and differentiation of a few cells but high enough to induce apoptosis in other cells. As consistent with the current understanding of staurosporine, the B35 and PC12 cells had shown higher cell counts. However the amount of cells which had differentiated was relatively lower than the RGC5 cells. It can be concluded that the staurosporine had been supporting the growth and differentiation of the cells which had survived the high concentrations as staurosporine had shown the largest percentage of cell differentiation, as shown in figure 1.


One limitation of this study was the time restraints that did not allow for enough data collection to perform quantifiable statistical analysis. Future studies could be undertaken in order to replicate this study on a larger scale so that enough data could be collected to determine if the results are statistically significant. Further studies could also investigate the effects of these growth factors on primary neuronal cells to see if the same patterns of results occur. Another future direction could be the use of apoptosis kits and resources such as Tunel imaging in order to test for markers of apoptosis and determine whether the low cell counts and zero cell counts are due to the cells undergoing apoptosis or necrosis.


The neuronal growth factors that were used all play a significant role in the growth and maintenance of cells within the human body. As previously described, all of these factors have differing but essential roles in various aspects of neuronal repair and protection which is why a comprehensive understanding of their modes of action and effects on different cell types is so important. NGF plays an important role in the repair, regeneration and protection of neurons, whilst BDNF mainly acts on neurons in the central and peripheral nervous systems, as well as areas that are related to memory and complex thinking such as the hippocampus. Retinoic acid is also essential in the patterning of neurons during the early stages of development. It can be seen that these growth factors, particularly NGF could play a role in the treatment of various neurodegenerative diseases such as Alzheimer’s disease. The growth factors effected different cell types in a variety of ways depending on concentration. Further study is required to evaluate a more concrete explanation of exactly how and if these growth factors could be used therapeutically.


This study aimed to investigate the effects of growth actors on the differentiation of various neuronal cell lines. In disagreement with our null hypothesis, it was found that the growth factors had varying effects on the different neuronal cell lines in terms of the cell counts, as well as the morphological features as the cells differentiated. This study also provided valuable research experience and an insight into the work of research groups within a cell biology lab, and provide experience to the methods and procedures that are used routinely.


References

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