Hi colleagues,
I'm studying the osteogenesis of MSCs. I used some cell lines before, so I don't have much experience with primary culture of MSCs.
Would you mind suggesting some protocols for isolating and inducing osteogenic differentiation of mouse MSCs?
I'm also confused about the sources of MSCs as some people use BM-MSCs, but some use calvarial osteoblasts. Which one shows better differentiation potential?
Thank you in advance.
By the way, there are some protocols suggested using heat-inactivated FBS, while some said that do not deactivate the serum. Could anyone give me some advice?
Esteemed Mr. Nguyen,
I attached articles with experimental protocols using DAG as osteogenic medium for MSC that may be useful to you.
Best regards from Brazil.
A frequent protocol used is here: http://www.ncbi.nlm.nih.gov/pubmed/19131962
Whether you want to use BM-MSCs or calvaria osteoblasts depends on your experiment. The former are mesenchymal stem cells and can differentiate towards multple lineages, the latter are osteoblasts already. The two cell type have different origins, since calvaria osteoblasts are periosteum derived.
@Igor: Thank you so much for your articles and the protocols. I'll try it for my experiments.
@Riccardo: Thank you for your advice. As I want to obtain a source of cells with high potential to differentiate into osteoblasts or osteoblast-like cells that express high level of osteoblastic markers (we use Runx2, Bglap, Spp1, Col1a1 and Col1a2 in our lab) over a 2 or 3-week culture period, I am confused between these 2 sources. I also used C3H10T1/2 to study osteogenesis before, but I could not obtained a proper population of osteoblasts after 3 weeks of differentiation, so I decided to use the primary culture. Currently, I plan to use knockout mice to see the relationship between our gene of interest and osteogenic process, so I need to see to what extent calvarial cells from knockout mice can undergo differentiation. However, as those cells are already osteoblasts, I'm confused if it is a good model for this purpose. I would appreciate if you share your experience with me.
if you are looking at osteogenesis, I would not use calvaria cells. Those are already osteoblasts that partially de-differentiate when you expand them in vitro and then re-differentiate when subject to osteo inducing factors. To study osteogenesis (of long bones) you want to use mesenchymal stem cells or embryonic cells which are more appropriate models for osteogenesis. If you are specifically interested in osteogenesis of the skull, you should use periosteum cells, since skull osteoblasts are derived from the periosteum. What did you use as osteogenic inducer? Just beta glycerophosphate or also growth factors?
Hi Riccardo,
I used 10mM Beta-glycerophosphate, 50uM ascorbic acid, 100nM Dexamethasone for C3H10T1/2 cells before, but I obtained adipocyte-like cells in the culture from day 10 of differentiation.
This time, I will try 10mM Beta-glycerophosphate and 50uM ascorbic acid only for calvarial cells, but as I said before, I'm quite worried about this model.
As we have time limit, we're still considering using BM-MSCs as most of the protocols show that in order to obtain a good population of MSCs without contaminated cells, we need to expand the MSCs population for at least 3-4 weeks. In general, how long does it take you to obtain cells that are ready for the differentiation? And do you use FACS to characterize them?Add your answer
Thank you!
I do not use FACS, I just do a tri-lineage differentiation test. It might take some time to get enough cells from the bone marrow of mice - it also depends on how many mice you sacrifice. Starting from a couple of mice, it might take a few weeks. If you need mouse MSCs those can be purchased so you might just buy some of those. Did you add serum to your media? you need serum too
Do you mean that after isolating BM-MSCs from mice, a purified population of MSCs can be obtained a couple weeks after the initiation of culture? What are the components of your osteogenic induction medium? And could you obtain many osteoblasts derived from BM-MSCs in your experiments?
Yes, I usually add 10% FBS, 2nM L-Glutamine and antibiotics to MEM (alpha modified - Sigma Aldrich).
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