We did in vitro differentiation of MSC cells into osteogenic and adipogenic lineage cells using STEMCELL kit. The protocol recommended to use culture-expanded human MSCs between passages 1 - 4 but cells of passage number 8 were induced to form osteoblast and adipocytes by culturing in osteogenic and adipogenic induction medium for 14 days, 24- well plate was seeded by 10000cell/cm2 and incubated for 48h to reach 80% confluent, then the media was replaced with complete MesenCult™ Osteogenic and adipogenic Differentiation and medium was changed every 2-3 days Osteogenic and adipogenic differentiation was visualized by staining with Alizarin Red S and Oil Red O staining, respectively. It seems that lipid accumulation happened for negative control (no differentiation media) and also the same pattern for osteogenic negative control.
I was wondering if someone had thought of a better and more time efficient solution.
Thanks
Dear Peyman Habibi,
Which type of MSCs you used? If it was primary culture, I strongly recommend you work only with the cells in early passages (1-4). Contrary to immortalized cell lines (like HeLa), primary cell cultures lose their properties with each passage.
Most probably the problem is that you used cells in the 8th passage. Still, osteogenic and adipogenic potentials usually decrease due to passage - so I would expect that you should get no differentiation at all instead of differentiation in control samples...
Have you tried to stain the cells at the start point? Are you sure that your MSCs culture is homogeneous?
We briefly touched on the problem of MSC heterogeneity in vitro in our recent review of mesenchymal cells osteogenic differentiation. Maybe it would be useful to you.
Article Osteogenic differentiation: a universal cell program of hete...
Hi Peyman Habibi , From the attached presentation, I see that you have a successful adipogenic and osteogenic assay, you have strong accumulation of lipid droplets consolidating into one place and strong dye deposition, similar in case of osteogenic differentiation.
Your negative control stain is background noise, non-specific deposition of particles stuck to the cells, not bound to adipocytes or osteocytes. You should wash them a couple more times to get rid of non-specific stain. 60% isopropanol for oil red O and ddH2O for Alizarin S will be helpful to clean your negative controls.
MSCs in negative control can be triggered by cross-contamination during media change, which happens sometimes but does not constitute to good differentiation.
To assess differentiation, you can extract the stain to quantify it via spectrophotometry which will give you more assurance that your assay worked.
Hi Peyman,
My name is Sampurna and I am a Product Manager at STEMCELL Technologies.
It would be great if you can provide some details about the upstream workflow that you used to generate the MSCs. These kits are verified for bone marrow, adipose, and PSC-derived MSCs. The adipogenic kit is also verified with Umbilical Cord-MSCs. Different culture lengths are required for different tissue sources. Additionally, the expansion medium that the cells were grown in can impact the differentiation potential, especially at later passages like you mentioned. We typically do not see adipogenic or osteogenic differentiation in the negative control well. So that is indicative that the cell source or expansion medium used is changing the properties of the MSCs.
I hope this helps! If you have any more questions, feel free to contact us at [email protected].
Kind regards,
Sampurna
Hi sorry to jump on your question but I am doing a similar experiment with MSCs but my cells are not differentiating do you have any tips? and do you mind sharing your staining protocols?
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