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Rationale

Discuss about the Impact of Glucocorticoids on the C2C12 cells’ Differentiation and Proliferation.

All the vertebrates have three main types of muscles that can be differentiated on the basis of their morphology and their roles in the body. The skeletal muscles enable the vertebrates to move from one geographical area to the other. Smooth muscles are lined with internal organs like blood supplying vessels. The third one is the cardiac muscle that is located within the heart. Its main role is to help in pumping of the blood thought the body thus enabling metabolic processes. Skeletal muscles account 40% of the body weight.

The skeletal muscle cells are the largest cells in the body. During the developmental stages the myoblast also know as precursor fuses to form a multinucleated skeletal muscle cells through a process known as myogenesis. It’s possible to study Myogenesis outside the body of the host using cell lines myoblast like C2C12 cell lines. C2C12 cells are easily proliferate under more concentrated serum conditions and differentiate but fusion can only take place under low serum state. Therefore, making it a good tool in tests that involve myogenesis, muscle biology and metabolism. The cell line starts to show myogenesis when it riches 100% confluency. But when transferred to differentiation medium, the cells quickly increase myogenesis and differentiation results to the formation of myotubes (Ricotti et al 2012)

The experiment started by setting up 2 well plates and allowed to proliferate for about 4 days and then allowed to differentiate for about 10 days. These were 2 well plates out of six. The remaining 4 well plates were set by the laboratory demonstrators. Two of the remaining well plates that contained C2C12 were seeded on the day 1 before of the Lab# 3. The other two wells in the culture containing C2C12 were seeded on day 4 and they represented Day 4-time point. The proliferation extent was accessed by comparing the C2C12 cells seed on day and day 4 (figure 2). In the case of more cells in day 4 than day 1 proved that the proliferation might have occurred. Differentiation was to be accessed by comparing the day 1 cell and day 4. In case there is different cells, that shows that the differentiation took place.

During 2-4 labs, effect of dexamethasone on C2C12 cell proliferation was evaluated. A control experiment was set for day1,2 and 14 (Figure 3) and each treated with dexamethasone. In order to access the effect of the dexamethasone on cell line proliferation, 100uM dexamethasone was incubated one of the 2-day 4 cell well plates. The number of cells in the control day 4 well was compared with the experiment treated day 4 well.

Results

The number of cell that were present in each of the cell wells of all the cultured plates during differentiation may affect the extent to which differentiation is attained. If a big number of cells recorded in a well at the beginning of differentiation stage, that shows the interaction of cells to form myotubes.

Proliferated and differentiated cells of C2C12 were then fixed using 10% neutral buffered formalin. Then they were stained using eosin and hematoxylin. The morphologies of these cells were then accessed by counting cell on the hemocytometer. The following cell count were observed from the secondary square of hemocytometer. These were the cells that took up the trypan blue in their cytoplasm and nucleus. From the outer corner of the hemocytometer, 7 cells were observed at the top and 3cells on top left. The secondary square at the outer border of the hemocytometer grid, the following number of cells were observed. Square# 2 had 4 clear cells, 6 blue cells. Square #3 1 clear cell, 2 blue cells. Square #4 3clear cells and 4clear cells.

The main objective of the lab was to study how to do immunocytochemistry on proliferation and differentiation of C2C12. Protein Proliferation cell nuclear antigen (PCNA) was also analyzed and the Myosin heavy chain (MHC) on the 3rd and 7th day. Glucocorticoid receptors localization in control and DMSO were assessed on the 3rd day. Immunocytochemistry was performed on fixed C2C12 cells from the 3rd and 7th day. Proliferation and differentiation was assessed on the two days consecutively in the experimental procedures described in 2nd and 3rd lab. One dish of the cells was fluorescently labeled for myosin heavy chain (MHC), Glucocorticoid or PCNA using secondary and primary antibodies. PCNA and (MHC) was assessed on third and seventh day using C2C12. Protein expression differed between differentiation and Proliferation of C2C12 Cells. Glucocorticoid was assessed on the 3rd day in control and C2C12 cell treated with dexamethasone. C2C12 cells treated with the dexamethasone were put inside dexamethasone for a period of 24 hours before fixed on the day 3. The time period allowed for dexamethasone initiated glucocorticoid receptor and its transfer to the nucleus.

To our own understanding, this is the first demonstration does not only induce proliferation and differentiation. Dexamethasone also causes hypertrophy in muscles depending on the differentiation stage at the point of application. The effect caused by dexamethasone is step-dependent. Dexamethasone observed at myoblast cause hypertrophy in muscles and enhances myotube differentiation as well.

Cell viability

Similar case has been observed when the myotube were treated using dexamethasone one of the glucocorticoids. The main objective this Lab test was to investigate the effect that would be caused by glucocorticoid in differentiation and proliferation processes of C2C12 cells. More emphasis focused on the dexamethasone which demonstrated the genes that were involved in the proliferation and differentiation. A vital thing that was investigated in the experiment was C2C12 cells with dexamethasone, PCNA and myosin heavy chain. This treatment aimed at the growth stimulation of the cells as well as to accelerate differentiation process. (Figure 1,2,3) shows that treating C2C12 cell with dexamethasone resulted to a significant high number of C2C12 cells proliferation. This observation is in agreement with what was recorded by (D'Angelo et al 2012) who described C2C12 cell growth and differentiation stimulation with glucocorticoids producing almost similar results. Again (Fujimoto et al 2012) showed the impact of glucocorticoid on the proliferation and differentiation of C2C12 cells. Some of the results obtained at different points of the day, showed that differentiation can be accelerated significantly when exposed to dexamethasone. The same results have been illustrated by (Goljanek et al 2012) in the experiment done to show the pivotal for C2C12 cells myoblast differentiation.

The hemocytometer was used in the counting of the cells according to (Riss et al 2016). The secondary square on the hemocytometer has got about 9 squares. Each of those square measure around one mm squared. The central part is the counting region on the hemocytometer. Only those cells lined on the two lines of the big square are suppost to be counted (Shenoy et al 2014). According to (Minetti et al 2014) the suspension must be diluted enough to prevent counting some of the cells a second time due to overlap. The purpose of using Trypan Blue was to differentiate between the blue cells (dead cells) from the clear cells (live cells). The staining method is also called the dye exclusion staining. The counting of cells done provided us with a % visibility cells of 40.74% and the cell density of 375000cell/ml (Figure5). The data was obtained using the light microscope where the magnification technique was to be applied in order to reach the desired cell type (Huri et al 2014). The cells in the chosen square were counted so that the total number of cells required was to be a significant statistical count (Alberts et al 2013).

Using immunocytochemistry to access C2C12 cell proteins

Myogenic index is termed as the number of nuclei that are contained in the cells that have more than nuclei divided by the total number of cell nuclei provided in the image taken. These images are then used to solve the myogenic index by finding the percentage number of nuclei in the myotubes using Jenner-Giemsa culture. The culture should be put in the differentiation media and incubated for five days. Giemsa G25 (Sigma Aldrich) is used to fix the cells and then three different photos are captured. The nuclei in the myotube and those in the cells are then counted in each photo. Myogenic index is then calculated by dividing the total number of nuclei in the cell by total number of nuclei in the myotubes. Myogenesis is the process being determined. Increase in the myogenic index shows accuracy in the experimental images whereby several nuclei are captured (Srikuea et al 2012)

C6orf32 gene is gene found in chromosome 6 and its expression multiples during the differentiation of the human fetal myoblast.  It is expressed both in non- myogenic and primary cells and is separated from the 18th week of human fetal development. Experiments on c2c12 murine myoblast cell revealed that cells without c60rf32 shows a myogenic differentiation defect while when it is expressed in excess, it accelerates the formation of filopodia. This gene plays a number of functioning like filopodia formation, cytoskeletal arrangement and promotion of myogenic cell differentiation. The gene c6orf32 is specialized to specific functions whereby the cells lacking this gene are unable to perform those functions and thus results to serial defects (Thrivikraman et al 2013)

Immunochemistry experimental design can be used in visualization of specific proteins. In the cells. In the experimental process, two antibodies are used in the targeting of a particular protein. These two antibodies are the secondary antibody and primary antibody. Secondary antibodies with fluorophore binds on the target and binds to the primary antibodies. Therefore, when one is performing immunochemistry, its important to use correct fixative. A secondary antibody must bind to primary antibodies other than non-specific antigen. Serum contains an array of the antibodies. Hence it should be blocked because it might block the binding of secondary and primary antibodies (Shi et al 2013).

References

D'Angelo, M. A., Gomez-Cavazos, J. S., Mei, A., Lackner, D. H., & Hetzer, M. W. (2012). A change in nuclear pore complex composition regulates cell differentiation. Developmental cell, 22(2), 446-458.

Discussion

Fujimoto, Y., Abematsu, M., Falk, A., Tsujimura, K., Sanosaka, T., Juliandi, B., ... & Nakashima, K. (2012). Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell?derived long?term self?renewing neuroepithelial?like stem cells. Stem Cells, 30(6), 1163-1173.

Goljanek-Whysall, K., Pais, H., Rathjen, T., Sweetman, D., Dalmay, T., & Münsterberg, A. (2012). Regulation of multiple target genes by miR-1 and miR-206 is pivotal for C2C12 myoblast differentiation. J Cell Sci, 125(15), 3590-3600.

Ricotti, L., Polini, A., Genchi, G. G., Ciofani, G., Iandolo, D., Vazao, H., ... & Pisignano, D. (2012). Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds. Biomedical Materials, 7(3), 035010

Srikuea, R., Zhang, X., Park-Sarge, O. K., & Esser, K. A. (2012). VDR and CYP27B1 are expressed in C2C12 cells and regenerating skeletal muscle: potential role in suppression of myoblast proliferation. American Journal of Physiology-Cell Physiology, 303(4), C396-C405.

Alberts, B., Bray, D., Hopkin, K., Johnson, A., Lewis, J., Raff, M., ... & Walter, P. (2013). Essential cell biology. Garland Science.

Thrivikraman, G., Mallik, P. K., & Basu, B. (2013). Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro. Biomaterials, 34(29), 7073-7085.

Shi, K., Lu, J., Zhao, Y., Wang, L., Li, J., Qi, B., ... & Ma, C. (2013). MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix. Bone, 55(2), 487-494.

Huri, P. Y., Wang, A., Spector, A. A., & Grayson, W. L. (2014). Multistage adipose-derived stem cell myogenesis: an experimental and modeling study. Cellular and Molecular Bioengineering, 7(4), 497-509.

Luo, W., Wu, H., Ye, Y., Li, Z., Hao, S., Kong, L., ... & Zhang, X. (2014). The transient expression of miR-203 and its inhibiting effects on skeletal muscle cell proliferation and differentiation. Cell death & disease, 5(7), e1347.

Minetti, G. C., Feige, J. N., Bombard, F., Heier, A., Morvan, F., Nürnberg, B., ... & Fornaro, M. (2014). Gαi2 signaling is required for skeletal muscle growth, regeneration, and satellite cell proliferation and differentiation. Molecular and cellular biology, 34(4), 619-630.

Shenoy, A., & Blelloch, R. H. (2014). Regulation of microRNA function in somatic stem cell proliferation and differentiation. Nature Reviews Molecular Cell Biology, 15(9), 565.

Riss, T. L., Moravec, R. A., Niles, A. L., Duellman, S., Benink, H. A., Worzella, T. J., & Minor, L. (2016). Cell viability assays. Madigan, M. T., Martinko, J. M., & Parker, J. (2017). Brock biology of microorganisms (Vol. 13). Pearson.

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