Hi, I am doing the Osteogenic differentiation experiment on the MSC cells line from human bone marrow. The passage is the 6th. I saw the adipogenesis differention starts (2-5%) from 7th day on the same passage of cells. So the potential of differentiation for cells should be fine.
The diff medium I am using is StemPro from Gibco. They recommended to use the Alkaline Phosphatase and Alazarin Red S staining for the Osteo diff cells. The tech support from Gibco told me that on the 2nd week after been treated, the cells should show 30%-80% by Alkaline Phosphatase staing. But by Alazarin Red S staining, the cells would show around 50% on the 4th week. I am confused.
1. If the MSC express some percentage of Osteo diff by itself, that should not be determined by the different methods of staining. Which stainging provide the accurate performance of Osteo diff?
2. If I applied the Alazarin Red S staining, when can I see the Osteo diff begin (1-5%) ?
Any advice would be appreciated. Thanks very much.
Good question. Please find my answers below:
Osteoblast differentiation markers are stage-specific (early stage markers-alkaline phosphatase (ALP) and late stage markers (matrix mineralization).
1. Alkaline phosphatase expression or activity alone can not indicate if the cells have differentiated into mature osteoblasts. There could be a basal expression of alkaline phosphatase in the MSCs that is further increased with commitment to osteoblast lineage. This induction can be seen in very early stages of differentiation (1 week) and it can be selectively induced by Wnt or BMP-2 treatment alone even in the absence of a mineralization media.
2. Mineralization is a characteristic of osteoblasts and alizarin red-S stains the calcium deposits that occurs during the mineralization process. As such, mineralization is salt formation between calcium deposits in the matrix embedded cells, a process that takes quite long time (4 weeks). Remember, first matrix has to be produced and the mineralization of the matrix not the cells indicate osteoblast maturation. It can be sometimes seen that cells treated for prolonged periods with the mineralization media can show calcium deposits that are not mineralized matrix nodules rather mineralized cells. This is not the true osteoblast mineralization rather this is only a mineralaized cell in which case can not be associated with differentiation-extracellular matrix mineralization.
Therefore, to conclude, one needs to assess all markers of mature osteobalsts (eg. matrix production or nodule formation, mineralization of matrix, osteocalcin & osteopontin secretion) in addition to the assessment of expression of other early markers such as runx2, ALP, and osterix.
Hello , Thanks for your questions , I propose refer to my article (The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with
Mesenchymal Stem Cells). If you have other question and you dont get your answer. please email to [email protected].
Best regard
I apologize for entering this discussion without an answer, but with a question. Could someone please provide me with images of cultured osteoblasts during each stage of differentiation. I isolated cells from the chicken femoral epiphysis and used alizarin red after the first passage. I need some comparison, even if it's for mammals.
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