Dear colleagues,
I'm needing your help with the protocol of differentiation of mesenchymal stem cells, once I pretty new in this field.
I isolated "MSCs" from dental pulp of deciduous teeth using MesenCult (StemCell) and plated 4000 cels/cm² at 6well plate and treated for 21 days with the following differentiation medium:
osteogenic - dmem high gluc, pen/strep, 10% fbs, 0,1 uM dexamethasone, 50 uM ascorbic acid, 2 mM bglicerolphosphate;
chondrogenic: dmem high gluc, p/s, 10% fbs, 0,1 uM dexamethasone, 1% ITS, 1 uM sodium pyruvate;
adipogenic: dmem high gluc, p/s, 10% sfb, 20 ug/mL human insulin, 500 uM IBMX, 100 uM indomethacin.
After 21 days I stained the cells with von kossa for osteogenic (1% silver nitrate for 1 h at UV light), toluidine blue for chondrogenic (1% toluidine blue for 30 min) and oil red o for adipogenic (0,05% oil red o for 1 h).
This protocol did not work and the photo is attached.
Can anyone help me this protocol?
Thanks in advance.
My experience is in BM-MSCs, and I'm using Miltenyi Biotec medium and protocol for cell differentiation. It might help you check the link
http://www.miltenyibiotec.com/~/media/Images/Products/Import/0009300/IM0009391.ashx
Look at this paper it can help you with osteogenic differentiation, also do some stem cells markers staining or FACS sorting to be sure that you are culturing dental pulp stem cells and not differentiated fibroblast.
https://www.researchgate.net/publication/236614886_Engineering_bone_tissue_using_human_dental_pulp_stem_cells_and_an_osteogenic_collagen-hydroxyapatite-poly_L-lactide-co-_-caprolactone_scaffold
Article Engineering bone tissue using human dental pulp stem cells a...
Experience showed that MSCs need the right Media for diffenrentiation (DMEM, or better Alpha MEM with addition of 10mM ß-Glycerophosphat, 300µM Ascorbate, Dexamethasone, 10% FCS). For osteogenic differentiation von Kossa staining can be used at late timepoints (try day 30 e.g.) because HAP production starts very late in differentiation. A good marker would be ALP-activity (from day 10 on) or Kollagen (day 20) production.
Your diff media compositions are too "light" :)
For instance you're not adding TGFbeta3 to your chondro diff media. I strongly suggest to applying pre-formulated commercial media (miltenyi, lifetech, lonza...). Especially if you're not sure of the differentiation potential of your starting cells. At the end, you will not save so much money with self-made media. ;)
Hi Bruna,
I would suggest you to add 1uM Dexamethasone in the adipogenic differentation medium. That works for me. In addition, it is important to wash thoroughly the wells with PBS and filter the Oil Red solution before every use to avoid the precipitates observed in your pictures.
In regard of osteogenic differentiation, I think low glucose DMEM is a better option, and you could increase the b-glicerophosphate concetration to 10mM. Alizarin Red staining is very useful to stain calcium deposition as well.
Good luck!
Hello Bruna,
Differentiation of DPSCs is very easy and as per Maria, I strongly recommend alizarin for osteogenic diff.
Please have a look at my published article which is available in my profile, "Effect of uncontrolled freezing on biological characterticks of dental stem cells". Even DPSCs can differentiate to ostocyte like cells within 7 days. I have used same concentrations as given in publication and it always worked.
Hope it will help you !
At first: did you check for MSC markers (CD44, CD105/endoglin, ets.) o make sure that you got indeed MSC?
If you have proper cells and drove those to differentiate into direction of adipogenesis, you might see tiny fat droplets within cells even without any staining.
Osteogenesis staining: Alizarin red is highly recommended: van Kossa can give pause-positives. Osteogenic medium: vitamin C is essential, beta-glycerophosphate is not nee before 8-10th day of differentiation.
One more thing: if you have the proper MSCs, you can also use a phenol red-free medium - in that case the dexamethazon is not needed any more - as all corticosteroids it's not a good additive...
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