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Current Students
Current Students
Bridgitte
Carita
Emily Y
Jessy
Mohsin
Naomi
Background
Background
One implication of studying myogenesis: certain muscle and cardiac complications are treated via myoblast transplantation. The potency of myoblasts relies on muscle cells that can differentiate properly. This implies that finding the right growth conditions in which myogenesis can be regulated is crucial to grow cells that can adapt to the host environment (Tanaka et al., 2011).
Myogenesis
Myogenesis
Myogenesis is the process of developing skeletal muscular tissue, wherein myoblasts develop into myotubes. Myoblasts are mononucleated, committed muscle cell precursors with a star-shaped morphology and tend to be evenly distributed across the growth surface. MyoD is a transcription factor and master regulator expressed during the beginning of myogenesis that arrests cell proliferation for differentiation to begin. Due to continuous growth and cell division, cell-to-cell contact increases; and when cells are at 100% confluency, cell fusion and differentiation are triggered. During this stage, m-Cadherin is vital for the fusion of myoblasts to form myotubes. As myotube development progresses further, they can be more clearly identified by defined characteristics, such as being multinucleated and having a tube-like appearance.
Biomarkers of myogenesis study
Biomarkers of myogenesis study
MyoD
MyoD
MyoD transcriptional factor is one of the myogenic regulatory factors (MRF) that determines cell identity before muscle cell differentiation (Berkes & Tapscott, 2005). In muscle cells, MRFs promote transcription of myogenic genes by binding to the enhancer and promoter sites, and eventually, induces cell differentiation (Berkes & Tapscott, 2005). Expression of the MyoD family was found to be essential for committing to the differentiation stage; research on the null mutation of MyoD has identified signs of delayed early stages of myogenic development (Berkes & Tapscott, 2005). Hence, MyoD transcriptional factor is crucial for muscle cell differentiation, as it plays an important role in initiating differentiation. Therefore, MyoD would be observed exhibiting up-regulation in the early stages of myogenesis.
M-Cadherin
M-Cadherin
m-Cadherin is most commonly found in developing muscle cells. Its main function is to aid in cell-to-cell adhesion (Hollnagel et al., 2002). Since cell confluency is one of the requirements for initiating cell differentiation and fusion in muscle cells, m-Cadherin plays an important role in fusing myoblasts to form myotubes (Hollnagel et al., 2002). When observed through a microscope, M-cadherin molecules in myoblast cells appear to be evenly spread out throughout the cells; whereas, in differentiated myotubes or myofibers, m-Cadherin appears to be clustered at specific parts of the cell membrane where the basal lamina is forming (Hollnagel et al., 2002). In normally developing muscle cells, we would expect to see M-cadherin localized along the membrane where fusion is occurring.
Inducing Differentiation in vitro
Inducing Differentiation in vitro
Serum Types
Serum Types
Fetal Bovine Serum (FBS)
Fetal Bovine Serum (FBS)
A rich source of growth factors (GF), is typically used in C2C12 cell culture at a 10% concentration as growth media (GM) to promote cell proliferation. Media containing 10% FBS is commonly used to promote cell growth in myoblast stages, some studies maintained cell culture in FBS, sometimes at different concentrations, throughout the course of myogenesis.
Horse Serum (HS)
Horse Serum (HS)
One of the most commonly used serums in differentiation media (DM) in C2C12 cell culture studies to induce differentiation by switching from GM with fetal serums to DM with adult serums. HS lack GFs and, thus, cell cultures supplemented with HS are often called serum deprived. The absence of GFs inhibits cell proliferation and activates myogenic genes to initiate cell differentiation. Although the suggested concentration for the differentiation media is 2% HS, a wide range of HS concentrations, from 2%, all the way up to 10%, have been employed across scientific literature.
ConFluency
ConFluency
Confluency can be defined as the percentage of cell coverage, wherein each cell is making cell-to-cell contact over the plating surface (Milipore Sigma, n.d.). This definition only applies to monolayer cell cultures. Other than GF deprivation, cell differentiation can also be induced through achieving cell-to-cell contact. C2C12 myoblast cells will continue proliferating until cell-to-cell contact is achieved by the majority of the population, which then triggers downstream signalling to initiate cell differentiation.
In literature, cell differentiation is often induced without achieving cell-to-cell contact (80%-90% confluency), but in some cases, confluency is achieved upon switching into DM to induce differentiation.
Project Rationale
Project Rationale
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.
Research Question
Research Question
How do confluency, differing serum types and concentrations impact the localization and mRNA expression of m-Cadherin and MyoD in C2C12 cells at the onset of differentiation?
In order to answer our research question our group has decided on MyoD and M-cadherin as the biomarkers for our study to observe myogenesis in vitro. DAPI will be used to ensure that our samples are actually progressing through the stages of myogenesis on schedule. MyoD is a master transcriptional factor of myogenesis, as previously described, and is highly upregulated at the early stages of muscle cell differentiation. Hence, we would expect to see the stable expression of MyoD localized within the nucleus throughout myogenesis. With m-Cadherin, we can observe cell-to-cell interactions and would expect the overall distribution of the protein to localize along the membranes where cell fusion occurs. Both analyses of MyoD and m-Cadherin will be done through immunocytochemistry to observe the localization of our study targets, as well as qPCR to quantify their expressions at different time points of myogenesis.
Outline of Methodology
Outline of Methodology
Cell Plating
Cell Plating
C2C12 cells were grown in 10% FBS growth media until 80% or 100% confluency, at which point, half the samples were switched to the experimental differentiation media (10% or 2% HS) to induce cell differentiation. Control samples were maintained in the 10% FBS growth media and induced to differentiate at cell-to-cell contact (100% confluency).
Sampling
Sampling
Fixation and Permeabilization
Fixation and Permeabilization
Cells were fixed and permeabilized for imaging at the Myoblast, Day 0 (early stages of cell fusion), Day 4 (early myotubes), Day 7, and Day 11 differentiation stages. Such arrangements were made to observe the process of cell differentiation in vitro.
Cell Harvest
Cell Harvest
On each sampling day, growth media was removed, and cells were washed and scraped in cold PBS. Cells were collected via centrifugation and stored in a -80˚C freezer until RNA isolation.
SAMPLE Preparation
SAMPLE Preparation
Immunocytochemistry
Immunocytochemistry
The MyoD and m-Cadherin antibodies and DAPI were used for staining the cells to visualize the progression of cell differentiation in various growth conditions. This part of the experiment is vital for observing the localization of biomarkers (MyoD, m-Cadherin) at different stages of differentiation.
Antibody Dilutions
MYOD Monoclonal Antibody (1:50), which targets MyoD
M-cadherin Polyclonal Rabbit (1:100), which targets m-Cadherin
DAPI (1:200), which targets the nucleus
Alexa Fluor 488 (1:200), which binds to M-cadherin Polyclonal Rabbit primary antibody
Alexa Fluor 555 (1:200), which binds to the MYOD Monoclonal Antibody
Immunostaining
Primary antibodies were applied and incubated for 1 hour at 37˚C and 5% CO2. Secondary antibodies were applied after washing and incubated at 37˚C and 5% CO2 for 30 minutes. Finally, DAPI was added, samples were incubated at room temperature, washed in distilled water, and mounted.
RNA Isolation
RNA Isolation
RNA isolation was performed according to the PureLink RNA Kit manual. RNA samples were stored in -80˚C freezer or used immediately for RNA assessments (i.e., 1% agarose gel electrophoresis, Qubit BR, NanoDrop).
Reverse Transcription (High Capacity RT kit)
Reverse Transcription (High Capacity RT kit)
RNA concentrations were determined based on the NanoDrop assessment. cDNA synthesis was performed according to the High Capacity RT Kit manual.
Visualization and Quantification
Visualization and Quantification
Fluorescence Microscopy
Fluorescence Microscopy
Imaging was done using the CellSens Imaging Software. All channels were viewed at maximum gain settings (18 dB). DAPI exposure was set to 10 ms, and FITC and TRITC exposure was set to 100 ms. Objective magnification was 60X.
qPCR and Expression Quantification
qPCR and Expression Quantification
qPCR was done with the SsoAdvanced SYBR Green Supermix, myoD, m-Cadherin and GAPDH primer sets. GAPDH primer set was used as a reference gene allowing for the normalization of myoD and m-Cadherin. qPCR was run in accordance to SsoAdvanced SYBR Green Supermix manual.
Condition Subgroups
Condition Subgroups
Summary of Results
Summary of Results
fluorescent imaging
fluorescent imaging
MYOD staining
MYOD staining
Myoblasts seen under TRITC channel
Myotubes seen under FITC channel
m-cadherin staining
m-cadherin staining
Myoblasts seen under FITC channel
Myotubes seen under FITC channel
Non-specific antibody bindings were observed across all samples.
MyoD staining:
We expected to only see nucleic structures under the TRITC channel, as MyoD is a transcriptional factor that should localize within the nuclei
However, membrane structures were observed under the TRITC channel, which was inconsistent with the literature and our hypothesis
M-Cadherin staining:
M-cadherin is a membrane protein that mediates cell-to-cell interactions, hence, was expected to localize along cell membranes
But nucleic structures were seen under the FITC channel
Conclusion: Both the MYOD Monoclonal Antibody and m-Cadherin Polyclonal Rabbit have non-specific antibody bindings; immunocytochemistry results are inconclusive.
Gene expression
Gene expression
m-Cadherin
m-Cadherin
Peaked on Day 4, decreased by Day 7
Highest Expression → 2% HS and 80% confluency
Control group has high variability (SD)
Overall lower expression when compared to MyoD
myoD
myoD
Peaked on Day 4, decreased by Day 7
Highest expression → 2% HS at 80% confluency
2% HS at 100% confluency: Peak MyoD expression on Day 7
10% HS at 100% confluency: overall lower expression than control group
Conclusion/Discussion
Conclusion/Discussion
Localization/Morphology
Localization/Morphology
The localization of MyoD and m-Cadherin is inconclusive due to non-specific antibody binding
Myoblasts appeared as mononuclear and star-shaped cells at the myoblast stage of differentiation
Myotubes appeared by day 7 for all experimental culture conditions and were thicker within the 2% HS at 80% confluency condition
Immunocytochemistry (ICC)
Immunocytochemistry (ICC)
Results regarding the localization of myoD and m-Cadherin were inconclusive due to non-specific binding of the antibodies used.
myoD localization was seen within cell nuclei (expected), but also in other parts of the cell (possibly, within the cytoplasm)
m-Cadherin localization was seen throughout the cells (also could be cytoplasmic), but was expected to localize along cell membranes
Gene Expression
Gene Expression
MyoD expression on control samples steadily increased up to day 4, then decreased afterwards
myoD expression is inconsistent with the literature
fusion appears on day 4 across samples
more information from ICC to make concrete conclusions
highest expression of myoD on day 4 with the 2% HS and 80% confluency conditions
m-Cadherin expression trend was expected
Expression increased up to day 4 and then decreased within all culture conditions
highest expression on day 4 with the 2% HS and 80% confluency
POssible Future Directions
POssible Future Directions
Repeat ICC with new antibodies
New differentiation conditions
10% HS vs 2% HS at 80% confluency
Repeat qPCR analysis again but with new sets of primers
New primers should have an efficiency of 90% to 110%
References
References
Berkes, C. A. & Tapscott, S. J. (2005). MyoD and the transcriptional control of myogenesis. Seminars in Cell & Developmental Biology. 16(4-5),585-595. https://doi.org/10.1016/j.semcdb.2005.07.006
Chowdhury, S. R., Muneyuki, Y., Takezawa, Y., Kino-oka, M., Saito, A., Sawa, Y., Taya, M. (2010). Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts. Journal of Bioscience and Bioengineering, 109(3), 310-313. http//doi.org/10.1016/j.jbiosc.2009.09.042
Hollnagel, A., Grund, C., Franke, W. W. & Arnold, H. (2002). The Cell Adhesion Molecule M-Cadherin Is Not Essential for Muscle Development and Regeneration. Molecular Cell Biology, 22(13), 4760-4770. https://doi.org/10.1128/MCB.22.13.4760-4770.2002
Tanaka, K., Sato, K., Yoshimda, T., Fukuda, T., Hanamura, K., Kojima, N., Shirao, T., Yanagawa, T. & Watanbe, H. (2011). Evidence for cell density affecting C2C12 myogenesis: possible regulation of myogenesis by cell-cell communication. Muscle & Nerve, 44(6), 968-977. https://doi-org.libproxy.mtroyal.ca/10.1002/mus.22224.
Check out our poster for Research Days Here: https://docs.google.com/presentation/d/1dMyXH6z2WGeT9-63lt3TPUDz4W1L6K54/edit?usp=sharing&ouid=108250243713628981832&rtpof=true&sd=true
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