Embedded Files
Table of Contents
Table of Contents
Lab Personnel
Lab Personnel
Micah W.
Senay W.
Henry K.
Background
Background
Image retrieved from: https://www.youtube.com/watch?v=8TZgf1zaVgU
Sarcomeres, Myosin, MHC4, and MYH4
Sarcomeres are known as the basic unit of contractile movement which are made up of myofibrils, consisting of 2 main protein filaments, the thick filament, myosin and, the thin filament, actin which are responsible for muscular contraction (Riddle et al., 1997). Myosin is a motor protein that derives chemical energy from hydrolysis of ATP into mechanical energy to create mechanical force for multiple motile processes (Cooper, 2000). Sarcomeres are found between any two Z-discs, in the myofibril, which are in a lined up sequence of approximately 100,000 sarcomeres per muscle cell (Riddle et al., 1997). A mature and fully differentiated muscle cell expresses highly localized myosin (Burattini et al., 2004) in its sarcomeres and is able to contract. For this reason, Myosin is considered a good indicator of differentiation and has previously been used as a marker of differentaition. Many types of isoforms are found when looking at myosin heavy chains. MHC4, myosin heavy chain 4, is a protein that is expressed by the MYH4 gene in sarcomeres (Feghali et al., 1992). MHC4 consists of the shape of a hockey stick that has a binding site, at the tip of the hockey stick, for actin filaments.
MYOGENESIS
Myogenesis refers to the process of development of skeletal muscle tissues where the progenitor myoblasts differentiate into myotubes, which later form muscle tissues. The myoblast is a muscle cell precursor that appears with a morphology representing a star-shaped, mono nucleated cell (Rochlin et al., 2010) that is observed to have an even distribution of a single layer of cells, when cultured, on growth medium . Myotubes are plurinucleate synctia that further differentaite to have the final morpho-functional characteristics of the muscle cells (Burattini et al., 2004).
Image retrieved from: https://www.sciencedirect.com/topics/engineering/myosin-heavy-chain
Conditions of Differentiation
Differentiation is a process where progenitor stem cells (myoblasts in our research) exit the cell cycle and develop into mature skeletal muscle cells. During C2C12 differentiation, individual cells fuse and elongate into multi-nucleated myotubes containing the contractile Sarcomeres made up of myosin (Rochlin et al, 2010).
There are two major ways to induce differentiation in C2C12 cells: cell to cell contact at 100% confluency using 10% Fetal Bovine Serum (FBS) culture media and deprivation of growth factors using Adult Serum. 10% FBS contains a wealth of growth factors, which promote proliferation until 100% confluency where differentiation is triggered by cell-cell contact (Tanaka et al., 2011). Adult Serums are lacking in growth factors, causing the promotion of proliferation to cease and for cells to begin the differentiation processes.
Research Question and Rational
Research Question and Rational
Question
Question
What is the effect of a lower serum concentration, 2% instead of 10% adult serum, in the induction of C2C12 myoblast differentiation by deprivation of growth factors compared to myogenic differentiation induced by cell-to-cell contact in 10% fetal serum?
Does the addition of laminin in the 2% HS condition influence myotube formation?
Rational
Rational
American type tissue culture (ATTC) suggests 2% adult horse serum to induce differentiation in C2C12 cells by deprivation of growth factors. 2% adult horse serum is also serum deprived compared to 10% FBS, the recommended concentration for induction of differentiation through cell to cell contact. The reduction in serum concentration might be a confounding factor that influences the formation of myotubes together with the deprivation of growth factors.
In our study, we compare 2% adult horse serum and 10% adult horse serum to observe the effects of lowering serum concentration on myogenic differentiation. Differentiation induced by cell to cell contact at 100% confluency in 10% FBS will serve as control. Laminin, an extracellular basement membrane protein, was added to 2% HS for a fourth condition, as another group in lab found that Laminin also had an effect on myotube growth speed (see the 2022/2023 Laminin Supplementation page on this website), and we set out to find out if this effect was additive with 2% HS.
Timeline Of Experiment
Timeline Of Experiment
Methodology
Methodology
Plating the cells
Plating the cells
C2C12 muscle cells were cultured in 10% FBS, with an abundance of growth factors, for cell proliferation to occur. Two of the samples had media changes to 2% horse serum (HS) and 10% HS both at 80% confluency to promote cell differentiation and inhibit cell proliferation. One sample was kept with the same differentiation conditions at 10% FBS, as a control, with cell-to-cell contact as the mode of differentiation. For Immunocytochemistry, cells were grown on coverslips placed in the dish. For gene expression, cells were plated directly onto the dish.
Sample techniques & Sample Preparation
Sample techniques & Sample Preparation
Obtaining Cells
On each day, outlined above, one coverslip was removed from the culture dishes for staining and fixating. On these days, media changes were done by aspirating the spent growth media and adding new media for further culturing. For gene expression, plates were scraped with a rubber scraper in sterile PBS, and centrifuged. The liquid was removed from this pellet, and the cells were stored at -80C until gene expression.
Staining, Fixating Samples, and Immunocytochemistry
C2C12 cells were stained and fixated for imaging the stages of cell differentiation. All samples were stained with MitoTracker (mitochondria staining) and fixated. Eight of the twelve samples were stained for myosin identification, with primary and secondary antibodies, excluding the four control samples that were only stained with secondary antibodies to determine if non-specific staining or auto fluorescence will be an issue during imaging. The last stain involved DAPI for nucleus staining. Imaging took place on day 0, day 4, day 7, and day 11 after cell differentiation has begun. Day 0 imaging presented early stages of cell fusion, day 4 imaging showed early stages of myotube formation, day 7 imaging showed late stage of cell fusion, and day 11 presented finalizing stages of cell fusion from myoblasts to myotubes.
Gene Expression
RNA was extracted and purified from cells using the PureLink RNA Minikit. To ensure RNA quality, extracted RNA was tested using Gel Electrophoresis and QUbit BR to determine purity and as close to an accurate concentration as possible. Once verified, cDNA synthesis was performed with a kit in order to perform Quantitative Polymerase Chain Reaction (qPCR) with myosin primers, and GAPDH as a reference gene. Once Cq values are acquired, the ΔΔCt method can be used to calculate the relative gene expression and give insights onto how much myosin is expressed in each stage of myogenesis.
Growth Conditions in vitro
Growth Conditions in vitro
10% Fetal Bovine Serum (100% confluency)
Fetal Bovine Serum (FBS) is used when differentiating cells at 100% confluency, as it is rich in growth factors (GF) compared to adult serums. GFs will promote proliferation, causing cells to stay in their growth cycle until differentiation is triggered by cell-to-cell contact. Some literature will use 10% FBS and wait until 100% confluency during cell culture, and some will use differing concentrations of Horse Serum (detailed below). It is currently unknown what effects these differing conditions have on the morphology of the cells themselves.
10% Horse Serum at (80% confluency)
Horse serum (HS) is a common reagent used in differentiating media when conducting research on C2C12 cells. This adult HS consists of limited growth factors (GFs) compared to FBS, therefore the cell cultures that are substituted with HS are commonly called serum deprived. Due to the lack of growth factors within HS, cell proliferation is inhibited and cell differentiation is promoted. Contemporary literature suggests to commonly use 2% HS, but other scientific literature has increased the concentration up to 10% HS. In all three trials, cell differentiation was induced at 80% confluency via switching culture media from 10% FBS to 10% HS.
2% Horse Serum at (80% confluency)
2% HS is the recommended concentration for differentiation by the inhibition of growth factors (ATTC). It is more diluted compared to the 10% HS, hence is more serum deprived. By changing the media to 2% HS at 80% confluency the cells are highly deprived of growth factors and are forced to start differentiating. To compare the impact of different concentrations of HS we have induced differentiation by 2% adult horse serum and compared it with 10% adult horse serum and our control, differentiation induced by cell-to-cell contact in 10% FBS.
Immunocytochemistry Images (2022)
Immunocytochemistry Images (2022)
For 10% FBS, nuclei are shown in yellow, myosin in green, and mitochondria in red. For 2% and 10% HS, nuclei are shown in blue, myosin is shown in green, and mitochondria are shown in red.
Phase Contrast Images (2022-2023)
Phase Contrast Images (2022-2023)
Scale bars contains large and small sections. The large lines indicate 0.1 millimeter in length where the small lines indicate 0.01 millimeter in length.
Myoblast
Top image on the left shows C2C12 cells cultured in 10% FBS and 2% HS on the right. Images on the bottom, left were C2C12 cells cultured in 10% HS and the images on the bottom right were C2C12 cells cultured in 2% HS + Laminin. C2C12 myoblasts are elongated, star-shaped cells with a relatively uniform cytoplasmic density. The cells are commonly found tightly packed and form a monolayer on the culture dish. The nuclei appear as dark, round structures located near the center of the cell. All four cultures in their respective culture conditions (10% FBS, 2% HS, 10% HS, 2% HS + Laminin) exhibited similar morphologies with star-shaped cell bodies and slightly elongated filopodia which attempt to reach adjacent cells to form stable contact between cells.
Day 0
Top image on the left shows C2C12 cells cultured in10% FBS and 2% HS on the right. Images on the bottom left were C2C12 cells cultured in 10% HS and the images on the bottom right were C2C12 cells cultured in 2% HS + Laminin. On day 0, after differentiation was induced with 10% adult horse serum (HS), the nuclei of the cells will be visible as small, dark, round structures located at the center of the each cell. The nuclei will be roughly the same size and shape for all cells, indicating that they are in an undifferentiated state. As the cells continue to culture and differentiate, they will begin to take on more distinct morphologies and structures. In both 2% HS and 2% HS + Laminin growth conditions the cells began to form cell to cell contact allowing for the next step of myogenesis, myoblast fusion. The nuclei in both of the 2% HS conditions are similar to those of 10% HS and the morphology is constant with a star-shaped cell body with a single nucleus found in the center of each cell body. The image at the top, 10% FBS control, shows similar morphology to the two other conditions with myoblasts present and contacting adjacent cells to initiate the process of myotube formation.
Day 4
Top image on the left shows C2C12 cells cultured in10% FBS and 2% HS on the right. Images on the bottom left were C2C12 cells cultured in 10% HS and the images on the bottom right were C2C12 cells cultured in 2% HS + Laminin. In 10% HS, C2C12 cells on day 4 become elongated and fused together to form multinucleated myotubes. The cytoplasm will appear granular, with distinct striations visible along the length of the myotubes. The nuclei of the cells will be visible as elongated structures located at the periphery of the myotubes, with multiple nuclei present in each myotube. The nuclei are flattened and elongated in shape, reflecting the shape of the myotubes. Myoblasts are still present in culture seen as star-shaped cells yet to fuse. A few myotubes are formed in the 2% HS condition at this stage. The morphology of the myotubes in the 2% HS codnition appears to be thinner, shorter and fewer in number compaed to the 2% HS + Laminin. In 2% HS + Laminin, similar myotubes are found with multiple nuclei visible in one elongated, spindle-link cell body. Myotubes begin to form striated patterns that begin to aligne themselves parallel to other myotubes. 10% FBS culture has not shown any observable changes in morphology from day 4. Myotube formation has not been observed and the myoblasts are still present in a mononucleated formation.
Day 7
Top image on the left shows C2C12 cells cultured in10% FBS and 2% HS on the right. Images on the bottom left were C2C12 cells cultured in 10% HS and the images on the bottom right were C2C12 cells cultured in 2% HS + Laminin. 10% HS cultured C2C12 myotubes on day 7 have become thicker and well-defined. The cytoplasm will appear dense and striated with the walls of the myotubes forming parallel to adjacent myotubes indicating that they are capable of contracting and generating force like skeletal muscle cells. The nuclei of the cells continue to appear elongated with multiple nuclei per myotube. Formation of multiple myotubes is observed in the 2% HS that look thinner compared to the other conditions. C2C12 cells cultured in 2% HS + Laminin showed similar morphology of myotubes, as in 10% HS, but the length of the myotubes is much longer, whereas the width of the myotubes is smaller compared to 10% HS. In both cultures, the presence of myoblasts has diminished, but not completely. C2C12 cell culture in 10% FBS has shown the first noticeable myotubes present in the culture. These initial myotubes have been observed on day 4 in growth conditions 10% HS and 2% HS + Laminin supporting our hypothesis of a higher rate of myotube formation is caused by deprivation of growth factors in the culture media.
Day 11
Top image on the left shows C2C12 cells cultured in10% FBS and 2% HS on the right. Image on the bottom, C2C12 cells cultured in 10% HS. In 10% HS and 2% HS, C2C12 myotubes on day 11 show growth progression in width and length of myotubes. Striated patterns of myotubes becomes evident with myotubes reaching lengths of 0.6-0.9mm in length and 0.2mm in width. The nuclei continue to appear elongated and flattened, reflecting the shape of the myotubes. Parallel patterns of myotubes is seen throughout the culture showing organization. Myoblasts are rarely seen in the culture as most of them fused into myotubes. In 10% FBS, as seen in the top image, is showing small in width and length myotubes that exhibit a striated pattern aligning themselves in parallel to adjacent myotubes.
Gene Expression Graphs
Gene Expression Graphs
Trial 1-3 Combined Normalized Gene Expression Graph
All samples start with very low myosin expression. By Day 4, non-laminin samples have had the gene switched on and are producing some myosin, but, in line with the microscopy images showing myotubes already present at day 4, the HS+laminin sample has a large jump in gene expression. By day 7, all samples show myotube and thus gene expression is noticeably heightened in all conditions. Laminin overall had a very high gene expression compared to other samples, suggesting that the HS + Laminin effects on myptube formation are additive, and that the presence of laminin may have some effect on myosin gene expression.
Future Direction
Future Direction
Based on the results that were gathered, it suggests that growing C2C12 cells in different growth conditions (2% HS + Laminin or 10% HS) will affect the process of myogenesis. In both, 10% HS and 2% HS + Laminin, myotube presence was noted on day 4, compared to the growth condition most commonly used in literature,10% FBS, where myotubes were only observed on day 7. Researchers should be vigilant in using specific growth conditions to ensure a reproducible experiment and to ensure the culture conditions are not affecting the rate of myogenesis in C2C12 cells.
POSTER
RELEVANT PUBLICATIONS
American Type Tissues Culture
https://www.atcc.org/products/crl-1772
Blau, H. (2022). C2c12 - crl-1772. ATTC. Retrieved from https://www.atcc.org/products/crl-1772
Burattini, S., Ferri, P., Battistelli, M., Curci, R., Luchetti, F., & Falcieri, E. (2009). C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. European Journal of Histochemistry, 48(3), 223–234. https://doi.org/10.4081/891
Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Actin, Myosin, and Cell Movement. Available from: https://www.ncbi.nlm.nih.gov/books/NBK9961/
Feghali R, Karsch-Mizrachi I, Leinwand LA, Kohtz DS. Four sarcomeric myosin heavy chain genes are expressed by human fetal skeletal muscle cells differentiating in culture. Gene Expr. 1992. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057361/
Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Section II, The Organization, Structure, and Function of Muscle. Available from: https://www.ncbi.nlm.nih.gov/books/NBK20168/
Rochlin K, Yu S, Roy S, Baylies MK. Myoblast fusion: when it takes more to make one. Dev Biol. 2010 May doi: 10.1016/j.ydbio.2009.10.024. Available from: https://pubmed.ncbi.nlm.nih.gov/19932206/
Tanaka, K., Sato, K., Yoshida, T., Fukuda, T., Hanamura, K., Kojima, N., Shirao, T., Yanagawa, T., Watanabe, H., 2011. Evidence for cell density affecting C2C12 myogenesis: Possible regulation of myogenesis by cell-cell communication. Muscle & Nerve 44, 968–977. doi:10.1002/mus.22224. Available from: https://doi-org.libproxy.mtroyal.ca/10.1002/mus.22224
Page updated
Report abuse