I currently use 2% HS to induce differentiation, as many people commonly do, and it works very well. What mechanistic quality of HS makes it better?
...I guess that there is no a mechanistic reason, probably the growth factors present in the HS are more suitable or exploitable by C2C12 receptors..You could assume that the serum coming from an horse which has a more important muscularity, if compared with bovine, might contain an higher proportion of factors which induce and/or mantain the differentiation from myoblast to myotubes.
I can't quote any specific reference, but I have always learned that it is because of the much lower content in growth factors of adult horse serum versus fetal bovine serum. A high growth factor content, plus supplementation with FGF2 keep the myoblasts proliferating and prevent differentiation, while loss of these signals allow progression into the differentiation parthway, as it happens spontaneously after in vivo implantation. I would check the woks of the Blau lab (Stanford) for more detailed information. Hope this helps.
HS is low in factors. Heat inactivated is best. FBS is rich in factors (as it is FETAL) when tissue expansion is occurring. I am not aware of a detailed study on this as much of the muscle culture approaches were developed before most of the growth factors were identified. In our lab, we often used define media as opposed to seras. Hope this helps.
Thank you... this satiates my curiosity. I could not find any specific references either, and assumed this was one of those situations where the "what" was defined before the "why."
C2C12 cells have 2 phases - proliferation and differentiation. During normal culture using FBS the cells are highly proliferative because FBS contains factors for proliferation. In order to differentiate C2C12 cells, you have to slow down the cells by withdrawing them from proliferation and switch to differentiation state. That is what you accomplish by supplementing the medium with adult horse serum instead of FBS. Also we use a much lower percentage of horse serum (2%) to ensure that proliferation is slowed down considerably. Some people use 0.1-0.2% FBS instead of horse serum, the ultimate purpose being the same.
Hi Nicholas,
I believe it is the higher Insulin content of the horse serum compared to FBS that makes it more suitable for differentiation of myogenic cells in addition to being lower in factors! :)
You can always supplement your 2% HS differentiation media with appropriate amount of Insulin (and Apotransferrin) for better differentiation of your myogenic cell cultures.
Best,
Mohsen
You will have to look at the 'original' papers by Konigsberg. Holtser and others back in the 60's and even 50's when they started growing muscle cells in vitro. I cannot recall the specific reference but it would be an interesting history lesson for you.
Start here and go backwards through his references
Buckley, P. A., & Konigsberg, I. R. (1977). Do myoblasts in vivo withdraw from the cell cycle? A reexamination. Proceedings of the National Academy of Sciences of the United States of America, 74(5), 2031–2035.
Yaffe and Saxel (1977) established the myogenic cell line and used a switch from 20%FBS to 10%HS after the cells became confluent. The idea was that FBS drives pluripotency and proliferation, but they did not define the factors.
http://www.ncbi.nlm.nih.gov/pubmed/563524
Reducing or removing serum slows or haults proliferation without additional supplements. Here is the reference that originally used defined conditions (bovine insulin and human transferrin) to induce C2C12 differentiation:
Changeux et al. 1986. Effects of chlorpromazine and phencyclidine on mouse C2 acetylcholine receptor kinetics.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1182877/
The low serum versus defined differentiation protocols have also been compared for differentiation via gene expression of myosin chains:
Cox et al. 1990. Transcriptional regulation of actin and myosin genes during differentiation of a mouse muscle cell line.
http://www.ncbi.nlm.nih.gov/pubmed/2201580
Just the difference between nutritional status does not explain why serum starvation alone fails to induce MHC, Myogenin, Caveolin-3 expressions compared to 2%HS. While screening for compounds capable of inducing myogenesis in C2C12 myoblasts we use serum starved cells treated with vehicle as vehicle-control cells and 2%HS containing cells as positive control of differentiation.I believe, as Mohsen stated above, insulin or any other yet unknown agent in HS might be a factor here.
The only reason Horse Serum is used is money. It is cheaper to used horse serum on cells that divide extremely fast.
FBS will work just fine.
I have used both HS (during my PhD) and FBS (now in my postdoc) for differentiating C2C12. Both work well, atleast for me. The only difference is that I used 5% HS compared to 0.2% FBS (now) for the differentiation.
@Ziwei Fu I don't use insulin to differentiate C2C12. The HS or FBS are sufficient and works well. All the differentiation associated markers are expressed well with this.
And yet, does anyone know the mechanism by which differentiation occurs? What triggers differentiation?
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