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Research question
How do C2C12 cells differentiate in an adult serum with reduced growth factors at 80% confluency compared to 10% fetal bovine serum (FBS)?
Background information/Project rationale
C2C12 cells are grown in 10% FBS. 80% confluent Day 0, Day 4, Day 7, and Day 11 cells are differentiated in 2% horse serum (HS) to compare the effects of the different serums. Researchers have used 10% FBS as ideal growth conditions and 2% HS as a popular choice for differentiation medium. However, there is ambiguity whether a an adult serum with low in growth factors (GF) is necessary for cell differentiation. In other words, what is the difference between induction of cell differentiation via cell-to-cell contact and reduction in GF?
Given the inconsistent use of cell-to-cell contact and reduction in GFs to induce cell differentiation across literature, the purpose of this project is to define the potential effects of cell confluency and growth factor reduction at the onset of C2C12 cell differentiation throughout myogenesis. It is also hoped that the findings of this study would contribute to establishing a concrete cell culture protocol within the discipline.
Phase Contrast Microscopy Images at 200X Total Magnification
10% FBS (control)
2% HS (experimental)
Myoblast
Day 0
Day 4
Day 7
Day 11
Both control and experimental conditions had myoblast formation and cells were mononuclear and star shaped. Within the experimental condition cells were closer together
By day 0 within both conditons cells appeared to be 100% confluent
At day 4 there was an increase in fusion between myoblasts and early development of myotubes
At day 7 it was evident that within the experimental condition there was increased myotube formation in comparison to the control condition
By day 11 both conditions had formation of myotubes with very little myoblast cells visible
Experimental condition has more myotubes
Myoblast
Day 0
Day 4
Day 7
Day 11
Immunocytochemistry imaging
C2C12 cells were cultured in DMEM with control media (10% FBS at 100% confluency). Immunofluorescence microscopy was done using an Olympus Inverted Microscope IX51 and cells were photographed with the Olympus cellSens Standard software at 600X magnification using 100ms exposure settings. MyoD was observed under the FITC channel and is critical in turning on muscle specific genes and m-cadherin was observed under the TRITC channel and mediates cell-to-cell adhesion allowing formation of myotubes. Non-specific binding was observed as membrane structures were seen under the TRITC channel and the nuclei was seen under the FITC channel.
Relative gene expression data
MyoD
Overall, MyoD has a higher expression in experimental 2% HS than in the control 10% FBS condition
MyoD has the highest expression in day 4 myotubes in both control and experimental conditions
Decreasing level expression further into differentiation
M-Cadherin
M-Cadherin had a similar expression trend as the MyoD group with a increase in relative gene expression until day 4 for the control and experimental groups
The control group had higher gene expression only at the day 0 stage
Expression was highest for the experimental condition at day 4
Discussion and Conclusion
Morphology and localization
Myoblasts appeared as mononuclear and star-shaped cells at the myoblast and day 0 stages of differentiation
By day 4 fusion was visible and the cells started to become multinucleated
Myotubes appeared by day 7 for all experimental culture conditions and were thicker within the experimental condition (2% HS at 80% confluency)
Immunocytochemistry
The localization of MyoD and m-Cadherin is inconclusive due to non-specific antibody binding
MyoD had fluorescent signals detected throughout the cell and m-Cadherin signals were detected within the nuclei
BLAST search revealed that the proteins receptor-type tyrosine-protein phosphatase V (XP_021018398.1) and protein transport protein Sec24D isoform X1 (XP_029393201.1) had alignment with the MYOD monoclonal antibody and the M-cadherin Polyclonal Rabbit had protein alignment with Fat4 (ABB88946.1)
Relative gene expression
Induction of differentiation in 2% HS at 80% confluency results in greater levels of gene expression than induction of differentiation at cell-to-cell contact (i.e., in 10% FBS)
Researchers studying myogenesis in vitro will need to take into account these differences in gene expression
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