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Cellular differentiation is the process by which an unspecialized cell develops into a specialized cell. Differentiation is important both in unicellular and multicellular organism. Differentiation dramatically alters the cell's shape, size, metabolic activity, and response to signals etc. In multicellular organism an embryonic cells can give rise to different type of cell through differentiation.
When embryonic cells are grown under proper conditions in culture, they remain unspecialized /undifferentiated. But when these cells are allowed to clump together to form embryoid bodies they start differentiation immediately. Differentiation can be activated by different type of cell signaling molecules such as Nerve Growth factor (NGF), which is an external signal molecule of neurotrophin family and act as a differentiation factor. Neurotrophin also promote functions other than differentiation that is, maintenance of survival, and regulation of metabolic activities . Neurotrophin bind to Trk family of the receptor tyrosine kinases and activates a number of intracellular signaling molecules via a ras-dependent pathway. As a result of neurotrophin prompt cell signaling intermediates, sensory and sympathetic neurons differentiate and avoid programmed cell death .
Tumor cells are often used for in-vitro studies of cells differentiation because of rapid cell division e.g. PC12 and COS7 cell line.
PC12 cell line is derived from a rat pheochromocytoma tumor cell line. These cells have cell surfaces receptor for NGF. When PC12 cells are exposed to NFG, they start differentiating and form a structure resemble to sympathetic neurons .Upon exposure to NGF, PC12 cells cease proliferation and start terminally differentiation and extend neuritis . It has been shown that NGF bind to receptor Trk-A, which present on the surface of PC12 cells, as a result Trk-A dimerize and transphoshorylate, which lead to activation of variety of signaling molecules for cell differentiation . These distinct biological responses to NGF made PC12 cells a model system for neuronal differentiation.
While COS7 cell line is derived from monkey kidney having feature of fibroblast. Unlike PC12, COS7 does not respond to NGF because of lack of cell surface receptor.
The purpose of this experiment is to study the effects of NGF on different cell lines in the presence and absence of MEK-inhibitor, by observing morphological difference of cell lines by adding rhodamine-phalloidin and staining dye DAPI for better visualization under fluorescent microscope.
Materials and methods
Plating the cells
For cell culturing cover slips were treated with 50μg/ml collagen in incubator for 30 minutes. Cells were rinsed with 1xPBS and detached by treating with 1xTrypsin/EDTA per dish for 5 min for PC12 and for 8 min for COS7. DMEM containing 2 mM L-glutamine, 1 mM Na-pyruvate, 1 mM Na-bicarbonate and 1x penicillin/streptomycin supplemented by 10% FBS for COS7 or 5% FBS and 5% HS for PC12 was added and cells were centrifuged at 1000rpm for 5min. Supernatant was aspirated and cells were diluted in order to have a confluency of about 70% for PC12 and 40% for COS7. Each cell suspension was transferred to respective wells containing collagenized cover slips and cultured over night.
Induction of differentiation
One well with PC12 and one with COS7 were treated with 20 μM MEK-Inhibitor for 30min at 37°C. Then wells with inhibitor and one more well with each culture were treated with 0.05μg/ml NGF and cultured over night.
Cells were rinsed with PBS, fixed with 4% paraformaldehyde for 15min at RT and rinsed with PBS again. After that cells were treated with permeabilisation solution containing 10% fetal bovine serum (FBS) and 0.1% Triton-X-100 in PBS for 5 minutes and rinsed with PBS. Cultures were stained in the dark for 30 minutes with staining solution containing 10%FBS, Rhodamine-Phalloidin 1:1000(v/v) and DAPI 1:1000(v/v) in PBS, rinsed with PBS and left in water. 6 μl of Mowiol/DABCO were put onto a labeled slide and covered with cover slips with attached cells so that cells ended up in between cover slip and slide. Then cover slips were sealed with nail polish.
Either untreated or treated with inhibitor and NGF PC12 cells had round shape (Figure 1. A). Some cells were attaches together. The PC12 cells treated with NGF undergone morphological changes and acquired neuron-like morphology: cell processes with developed cytoskeleton (Figure 1. B).
Figure1. The schematic view of untreated or treated with MEK inhibitor and NGF PC12 cells (A) and treated with NGF (B).1-nucleus, 2-actin filaments.
Untreated COS7 cells had round shape. They were not affected by treatment with MEK inhibitor and NGF or NGF alone. After treatment cells had same morphology: round shape, no processes (Figure 2).
Figure-2. The schematic view of untreated, treated with MEK inhibitor and NGF, and treated with NGF alone COS7 culture. 1-nucleus, 2-actin filaments.
COS7 and PC12 cells have different origin and this is the major reason for different response to growth factors. PC12 cells come from adrenal medulla which originates from neural crest. Neural origination can be a possible explanation for the ability of PC12 cell culture to respond to NGF by changing the morphology of the cells. Untreated cultures showed no morphological changes, as PC12 are adrenal cells and in absence of induction maintain their normal round-like morphology. Treated with MEK inhibitor and NGF cells also do not change their architecture due to the inhibition of key kinase in the cascade, triggered by NGF. Cells treated with NGF alone undergone morphological changes and acquired dendrite-like processes probably due to their common with neurons origin and respectively - ability to respond to NGF.
The COS7 cells on contrary in all cases showed the same morphology. This cell culture comes from kidney cells, which have mesodermal origin, and thus has nothing in common with nerve cells. Non-neuronal origin is a possible explanation of COS7 culture not responding to NGF treatment. There also was no response to inhibitor treatment probably because NGF didn't trigger any signal pathway in COS7 cells so there was no target for the inhibitor.