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Bridgitte Garcia & Naomi Oviahon
Research Question & Rationale
10% Fetal Bovine Serum (FBS) is widely used by researchers studying myogenesis, while 10% Horse Serum (HS) enhances myotube formation. M-cadherin is more prominent on Days 4 and 7, and myoD is best seen during the myoblast and Day 0 stages. If the localization of these proteins and cell morphology resulting from 2% HS is contrary to previous findings, the use of the recommended serum (10% HS) would be verified.
Research Question: Does applying 2% HS at 100% confluency allow the differentiation of myoblasts into myotubes to be studied in a more accurate environment?
Phase Contrast Images of Cells
Figure 1. Trial 3 C2C12 myoblasts at day -1 of myogenesis (80% confluency), grown in 10% FBS, Dulbecco's Modified Eagle Medium, and 1% Pen/Strep. The cells are quite spread out, and take on a star-shaped morphology, as they reach out to each other to make contact. Maximum contact is one of the 2 ways to induce myogenesis. This image was taken with a mobile device at 100x objective magnification. The phase contrast microscope was an Olympus CKX31.
Figure 2. Trial 3 C2C12 day 0 myoblasts (100% confluency), grown in 10% FBS, Dulbecco's Modified Eagle Medium, and 1% Pen/Strep. Having achieved maximum contact, the more cube-shaped cells have most likely begun myogenesis. At this stage, the media was switched from 10% FBS to 2% HS, to reduce growth factors and induce differentiation. This image was taken with a mobile device at 100x objective magnification. The phase contrast microscope was an Olympus CKX31.
Figure 3. C2C12 cells grown in 2% HS, DMEM, and 1% pen/strep. Day 4 of differentiation constitutes cells of a mixed population of myoblasts and myotubes. Cell fusion of myoblasts has begun, therefore some cells are multinucleated and elongated, while some remain as mononucleated and square-shaped myotubes. Image was contained by mobile device and Olympus CKX31 at the 100x magnetification.
Figure 4. C2C12 cells grown in 2% HS, DMEM, and 1% pen/strep. On day 7 of differentiation, the population of cells are mainly myotubes. Multiple myoblasts have fused together to make one single myotube. These cells are elongated in shape, and contain multiple nuclei in them. Myotubes in day 7 are typically larger in size compared to myotubes found in day 4. Image was obtained by mobile device and Olympus CKX31 at the 100X magnetification.
Immunocytochemistry and Fluorescence Microscopy
Negative Control: Myoblast Sample
Figure 5. Myoblast cells before day 0 of myogenesis at 80% confluency were taken with immunofluorescence and differential interference contrast (DIC) microscopy at 600x magnification. The nuclei of the myoblasts are coloured in blue, while myoD is coloured in green and m-Cadherin is coloured in red. There is non-specific binding for myoD and m-Cadherin, therefore, there are no specific areas that are green or red (this is expected for the negative control sample because no primary antibodies were applied during the staining procedure).
Control Sample: Day 0
Figure 6. Myoblast cells at day 0 of myogenesis with 100% confluency, images were taken with immunofluorescence microscopy at 600x magnification. MyoD is present in early stages of myogenesis and can be observed as green in this image. Differentiation is just beginning, and cell fusion has not occurred yet, hence, the mononucleated cells. The nuclei are round and separated.
Experimental Samples: Days 4 & 7
Figure 7. On Day 4 of myogenesis, the cell population in the image is mixed; some have yet to initiate fusion, and others have done so already.
Figure 8. Myogenesis Day 7 makes for a more eventful scene, with many nuclei gathering to form separate myotubes, stretching along with them in an attempt to conform to the cell's new shape.
Relative Gene Expression Data
Figure 9. Relative gene expression of myoD in relation to the reference gene, GAPDH. Samples grown at 10% FBS is labelled blue, while 2% HS is labelled as orange. MyoD expression in cells grown in 10% FBS steadily increases from the MB stage up until day 4, where it is expressed the highest. In cells grown in 2% HS, myoD increased after each day, with the highest expression on day 7.
Figure 10. Relative gene expression of m-cadherin, normalized to the reference gene, GAPDH. Cells grown in 10% FBS (control group) give data in blue; 2% HS at 100% confluency conditions (experimental group) in orange. Control group data was widely variable, and shows a sudden drop in expression on day 7. In contrast, there is very little variability in the experimental group, which also shows a steady increase in expression, and a smaller decrease at day 7.
Conclusions
Morphological Differences
Overall, changes in cell morphology, indicating progress through myogenesis, were successfully observed in both control and experimental groups
Larger myotubes could be seen in samples growing in 2% HS (at 100% confluency), indicating the positive effects of 2% HS on myogenesis (at least, in terms of cell structure)
Immunocytochemistry (ICC)
Unfortunately, non-specific binding of antibodies was identified during Trial 2 of antibody testing
unusual fluorescent signals seen on incorrect channels (fluorescence microscopy)
BLAST results revealed multiple other proteins that antibodies could have bound to
Further investigation of these growth conditions (2% HS at 100% confluency) vs control conditions (10% FBS), and their effects on the localization of myoD and m-cadherin, is required
Relative Gene Expression
MyoD
C2C12 cells that were grown in 10% FBS (control) had an increased expression of myoD from the myoblast (MB) stage up to day 4. There is a dramatic decrease of myoD expression from day 4 to day 7. Cells grown in 2% HS (experimental) lacked growth factors found in FBS, and the expression of myoD is steadily increased throughout each sample day. MyoD had a higher expression when grown in experimental conditions on MB, day 0, and day 7 when compared to cells grown in control conditions. This suggests that a lack of growth factors increases myoD expression. On day 4, the myoD expression in control is higher than in experimental, which suggests that the abundance of growth factors increased myoD expression.
M-Cadherin
Cells that were grown in both growth conditions (10% FBS, control; 2% HS, experimental) had increased expression m-Cadherin throughout each day and drops on day 7. The control conditions with the abundance of growth factors resulted in lower expression of m-Cadherin when compared to experimental conditions.
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