I have been trying to differentiate umbilical cord MSCs (from wharton's jelly) to osteogenic lineage but I have failedmany times, I have been using the classic cocktail composed by Dexamethasone, Ascorbate and B-glycerophosphate in 10% FBS DMEM high glucose . Are there critical suggestions about cell confluence, ingredients preparation, or anything that I need to know about this?
Many thanks
Hi Francisco,
The attached paper describes the trilineage differentiation of UC-MSCs. Check to see if the concentrations of the components of the osteogenic cocktail are the same as you are using. Also, they use DMEM low glucose and you mentioned you are using high glucose.
Good Luck!
http://deepblue.lib.umich.edu/bitstream/handle/2027.42/85116/ten_tea_2009_0706.pdf?sequence=1
Try this? It's the same cocktail as your with Vit D added. Maybe it might work.
Dear Francisco
The problem is might be due to high glucose concentration (4.5g/L). Since high glucose inhibits the osteogenic differentiation (check this http://www.ncbi.nlm.nih.gov/pubmed/19949837 , this http://www.excli.de/vol12/hongchen_27062013_proof.pdf and there are many other references)
Differentiation is good if you use low glucose (1g/L) (i have used normal glucose (3.5g/l) some time but the results were not good like LG).
There is no problem with classic composition, it done well for all (reported this composition is being used for IPSCs as well).
Check the following protocol. It worked well for us.
http://www.bio-protocol.org/e308
Crucial item in osteogenic differentiation is beta glycerophosphate. So store it in proper condition. if its old consider to get a new one.
Note- High glucose is recommended for chondro and adipo differentiation not for osteo.
all the best.
Dear Fransisco,
I used Stem pro osteogenesis kit from Gibco (cat. no.A10072-01), with good result. The calcification seemed thicker compared to BM MSC or Adipose MSC.
However, for adipogenic differentiation using Stem pro adipogenesis kit, showed very tiny lipid droplets, compared to much larger droplets in BM MSC or Adipose MSC.
Jeanne
Hello
i agree with all researchers and suggest use medium Eagle (α-MEM)
For that you can follow this protocol :
1-Incubate cells in α-MEM medium for three or four days. When over 80% confluence was reached.
2- Medium replaced by an osteogenic induction medium (OIM) containing 1 nM dexamethasone, 20 mM beta-glycerophosphate, and 50 μM ascorbic acid.
3- MSCs treate for two weeks. and , The osteogenic differentiation evaluate by real time qRT-PCR after 11 days
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131277/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370627/
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