Study Of Myogenesis And Proliferation Of C2C12 Cells

The general aim of the experiment was to study myogenesis of C2C12 cells and investigate the appearance of Myosin Heavy Chain and proliferating cell nuclear antigen (PCNA) during differentiation and proliferation. This aim was achieved through several lab sections with different objectives. The first lab section’s aim was to learn how to seed, culture and even differentiate and determine viability of C2C12 cells. Through the second lab section, preserving and staining of these cells was learnt to enable assessment of structural changes that take place during proliferation and differentiation. Additionally, the experiment sought to examine the effect of dexamethasone on proliferation and differentiation of C2C12 cells. In the last lab section, the objective was to learn how to perform immunocytochemistry on differentiating and proliferating C2C12 cells.

Myogenesis is the process through which new muscles are created (Hart, et al 2015) . A number of activities occur during myogenesis of skeletal muscles. Firstly, myoblasts are formed from muscle cell lineage; they proliferate and group themselves together within muscle development areas (Panda, et al 2016). Myoblasts then lose their proliferating ability and they start undergoing differentiation. As a result, these cells become elongated. The resulting elongated myoblasts then fuse together and myotubes, which are multinucleated, result. Lastly, maturation of myotubes occurs and as a result, skeletal muscle cells are formed (Panda, et al 2016).. The experiment was conducted using microscopy and immunocytochemistry techniques.

C2C12 refers to an immortalized myoblast cell line of a mouse and can be used to study myogenesis in vitro (Jgryall, 2013). This is made possible by culturing them in Dulbecco’s Modified Eagle’s Medium (DMEM), which can either induce proliferation or differentiation, depending on the growth factors in this culture medium (Ohta, et al 2014). When DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin and 2% amino acid is provided, C2C12 cells proliferate and when 2% adult horse serum, 1% penicillin/streptomycin, and 2% amino acids are provided differentiation occurs (Ohta, et al 2014). After differentiation, C2C12 cells lose their spindle shape and elongate. The elongated cells then fuse together and myotubes result, which later mature to become skeletal muscles.

Immunocytochemistry is an experimental technique used to visualize particular proteins in cells, and can either be direct or indirect (Kim, et al 2014).

Trypan blue exclusion was used in the experiment to determine the viability of cells. Some cells appeared blue and others appeared white when observed using a hemocytometer. The blue cells represented the unviable cells while the white cells represented the viable cells. Blue cells indicated that Trypan blue solution had penetrated the cell membrane of these cells and entered the cells’ cytosol, which only means that the cell membrane was broken apart, rendering the cells unviable. On the other hand, white cells were an indication that their cell membrane was healthy, not injured or broken apart, and therefore was properly functioning. It prevented entry of the Trypan blue solution into the cell’s cytosol, a clear indication that these cells were viable. Percentage viability of cells was calculated by the following formula and was found to be 80.58%

Results

The cell density was calculated for the undiluted sample of cryopreserved cells using the following formula and was found to be 207500 cells/mL.

Results for the cultured cells indicated that proliferation had occurred. This is demonstrated in day 4’s results represented in figure 2A in comparison to figure 1A. In figure 2A, the cells had increased in number, an indication that they had proliferated but they retained their spindle shape. More cells could be seen in the plate. In day 14, further changes in cells were depicted. As indicated in figure 3A, the cells were no longer spindle shaped, but had acquired an elongated shape. Additionally, more than one nucleus could be seen in the cells. Figure 3B was the control. Results observed in day 14 indicate that differentiation had occurred and as a result, myotubes had formed, accounting for the change in shape of the cells. The change in culture medium explains the different results in day 4 and day 14.

In regards to effects of dexamethasone, Increased number of cells in figure 5D as compared to 5A shows that dexamethasone increased proliferation rate when DNA was labeled using DAPI. In figure 5E, Glucocorticoid receptor had entered the nucleus of the cells and attached to DNA cells, indicating presence of a dexamethasone. As a result the cells appeared individually, unlike in 4B, the control. In the merged image both cells labeled with DAPI and Glucocorticoid appeared individually in presence of dexamethasone (5f) while in 5C (control) only DAPI labeled cells appeared individually.

On day 10 and 4, Myosin Heavy Chain (MHC) and Proliferating Cell Nuclear Antigen (PCNA) expression was evident as seen in figure 6. On day 10, MHC was present and differentiation of C2C12 cells had started. On day 10, the C2C12 cells had undergone changes in their shape, becoming elongated, an indication that myotubes had been formed during differentiation. In day 4, more individual cells would be seen, in comparison to day 10, where only a few of the individual cells would be seen (figure6). For PCNA, it was expressed on day 4 (figure 7B).  On the fourth day the PCNA cell samples appeared to have been oval in shape, many cells appearing individually. On the 10^th day the PCNA were not expressed, indicating that proliferation was not taking place. High levels of PCNA were indicated on the 4th day (7B).

Trypan blue solution is usually used experimentally to demonstrate viability of cells, where viable cells are differentiated with unviable cells depending on the color they will stain (Hi Media Laboratories, 2010).  As Hi Media Laboratories (2010) states, normal cells have intact cell membranes while dead cells have lysed cell membranes. Due to ruptured or weak cell membranes, some cells absorb trypan blue, indicating that they are not viable. On the other hand, due to healthy cell membranes that can appropriately and selectively select substances in and out of the cell membranes, viable cells do not absorb trypan blue solution, accounting for their unstained, clear appearance. A hemocytometry is used to determine the number of viable and unviable cells in a sample and has been utilized in many studies (Zhao, et al 2015).

Discussion

Development of skeletal muscles is a process that is tightly regulated and involves mesodermal precursor specification into myoblasts followed by subsequent differentiation as well as fusion of these cells into myotubes (Deng, 2011). The current experiment was a demonstration of this sequential process, although in vivo. This is evident as observed in the changes in number and shape of the cultured C2C12 cells from the beginning to the end of the experiment on day 14. Different stages of myogenesis were induced by provision of the necessary medium and the effects of different substances on myogenesis as well was observed by introduction of these substannces into the culture.

For instance, proliferation of C2C12 cells was triggered by provision of DMEM solution containing 10% fetal bovine serum, 1% penicillin/streptomycin and 2% amino acid. This is the appropriate constituents of medium suitable to trigger and support proliferation (Jgryall, 2013). To induce differentiation of C2C12 cells, on the other hand, 2% adult horse serum, 1% penicillin/streptomycin, and 2% amino acids are the required constituents of DMEM medium  (Jgryall, 2013). This explains why during the first few days the DMEM added in the sample contained 10% fetal bovine serum, 1% penicillin/streptomycin and 2% amino acid and later a DMEM medium containing 2% adult horse serum, 1% penicillin/streptomycin, and 2% amino acids was added. As already mentioned, the medium added during the first few days allowed cell proliferation while the one added on day 10 triggered and supported differentiation.

According to Syverud, VanDusen, & Larkin (2016), dexamethasone is a glucocorticoid that triggers differentiation of myoblasts and fusion to form myotubes. On a study investigating the effect of dexamethasone on tissue engineered skeletal muscles, Syverud, VanDusen, & Larkin (2016) found out that dexamethasone increased the rate of differentiation of the tissue engineered skeletal muscles as well as satellite cells. This collaborates with the results observed in this experimental study, whose findings indicated a reduction in the rates of both differentaiation and proliferation of C2C12 cells in presence of dexamethasone in the culture. The effects of dexamethasone on the proliferating and differentiating C2C12 observed in the current study are in contrary with those observed by Pas, Jong, & Verburg (2000).  Pas, Jong, & Verburg (2000) found out that glucocorticoid inhibited proliferation and differentiation of C2C12 cells. The difference in the observed results may be associated with contamination of cultures during the experemental process.

Myogenic index refers to the ratio of the number of nucleus residing in cells that contain more than two nuclei to the total number of nuclei in C2C12 myotubes stained with hematoxylin (Lee, et al., 2010). The index identifys a quantitative value of differentiation, thus determining the differentiation extent (Veliça & Bunce, 2011). Calcium regulation is a cellular process that can be assessed by measurement of myogenic index.

H19 gene, just like the MHC gene, is upregulated during C2C12 cell differentiation (Milligan, et al., 2000). Evidence of expression of this gene in high amounts is suggested by appearance of specific patterns in the differentiating C2C12 cells (Brown, Parr, & Brameld, 2012). H19 gene helps in promotion of skeletal muscle differentiation (Dey, Pfeifer, & Dutta, 2014). It makes the C2C12 cells more specialized in carrying out differentiation during myogenesis by producing microRNAs miR-675-5p and miR-675-3p (Dey, Pfeifer, & Dutta, 2014).

Blocking serum is utilized during immunocytochemsitry to prevent non-specific antigens from particular antibodies, thus increasing binding of primary and secondary antibodies (Zhou, et al 2014). If only a mouse serum was accessible, the secondary antibodies would be derived from the mouse species and it is possible to label fluorescently two different proteins in the same cell using immunocytochemistry technique.

References

Brown, •. M., Parr, T., & Brameld, J. M. (2012). Myosin heavy chain mRNA isoforms are expressed in two distinct cohorts during C2C12 myogenesis. Journal of Muscle Research and Cell Motility , 32(6):383-390.

Deng, X. (2011). Skeletal Muscles Differentiation and Fusion are Regulated by the BAR-Containing PhoGTPase-Activating Protein (Rho-GAP). Journal of Biological Chemistry , 286, 25903-25921.

Dey, B. K., Pfeifer, K., & Dutta, A. (2014). The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration . Genes and Development Journal , 28(4): 491-501 .

Hart, C. A., Tsui, J., Khan, K., Ho, T. K., Shiwen, X., Ponticos, M., ... & Baker, D. M. (2015). Critical Limb Ischaemia in Adult Human Skeletal Muscle Increases Satellite Cell Proliferation but Not Differentiation. Surgical Science, 6(5), 198.

Jgryall. (2013). Research in Myogenesis: Working with the C2C12 Cell Line .

Kim, H. S., Kim, J., Jo, Y., Jeon, D., & Cho, Y. S. (2014). Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors. Stem cell research, 12(1), 60-68.

Laboratories, H. M. (2010). Trypan Blue: Cell Culture Tested. 1-2.

Lee, C. S., Yi, J.-S., Jung, S.-Y., Kim, B.-W., Lee, N.-R., Choo, H.-J., et al. (2010). Cell Death and Differentiation . Molecular Cell Biology , 17, 1254–1265.

Milligan, L., Antoine, E., Bisbal, C., Weber, M., Brunel, C., Forné, T., et al. (2000). H19 Gene Expression is Up-regulated Exclusively by Stabilization of the RNA During Muscle Cell Differentiation. Oncogene Journal , 19(50): 5810-5816.

Ohta, K., Ogawa, T., Kaise, A., Oda, A., & Endo, Y. (2014). Aliphatic substitution of o-carboranyl phenols enhances estrogen receptor beta selectivity. Chemical and Pharmaceutical Bulletin, 62(4), 386-391.

Panda, A. C., Abdelmohsen, K., Martindale, J. L., Di Germanio, C., Yang, X., Grammatikakis, I., ... & De, S. (2016). Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis. Nucleic acids research, 44(5), 2393-2408.

Pas, M. F., Jong, P. R., & Verburg, F. J. (2000). Glucocorticoid Inhibition of C2 Proliferation Rate and Differentiation Capacity in Relation to mRNA levels of the MRF Gene Family. Molecular Biology Reports , 27(2): 87-98.

Syverud, B. C., VanDusen, K. W., & Larkin, L. M. (2016). Effects of Dexamethasone on Satellite Cells and Tissue Engineered Skeletal Muscle Units . PubMed , 22(6): 480-489.

Veliça, P., & Bunce, C. M. (2011). A Quick, Simple and Unbiased Method to Quantify C2C12 Myogenic Differentiation. Muscle and Nerve Journal , 44(3):366-370.

Zhao, Z., Mahmood, A., Patra, P., Rho, J., & Macwan, I. (2015). Image processing tool for the Microorganism cell counting and its recognition.

Zhou, X., Li, L., Su, J., & Zhang, G. (2014). Decreased miR-204 in H. pylori-associated gastric cancer promotes cancer cell proliferation and invasion by targeting SOX4. PLoS One, 9(7), e101457