Hi. I wish to start attempting to isolate and experiment with Mesenchymal Stem Cells from human colon samples. However, it seems that all the procedures I come across are essentially the same as when our laboratory isolates fibroblasts. Being that fibroblasts have some limited differentiation potential and MSCs have been observed to take on the characteristics of fibroblasts in culture, I was hoping someone in the community could identify some methods that worked for them in a way where they absolutely verified they isolated mostly MSCs.
MSCs have specific CD profile. This would make this useful for the use in cell sorting. so I guess this is the method that I would recommend.
in the bone marrow sample it is easy to isolate these cells because they were the only cells that adhere to plastic. but since epithelial cells in colon samples adhere to plastic as well I would guess cell sorting would your best way.
then you can verify them by probing with MSCs markers. there is a minimal panel to confirm MSCs. I would use that.
As Arwa says isolating MSCs from tissues that contain a range of adherent cell populations is a lot harder than isolating from BM where the only adherent cells that will grow in standard media are MSCs and macrophages. I'd try to use FACS if you have the capability- there are a range of well validated markers for MSC that should enable you to sort a reasonably pure population (PDGFr, STRO-1 etc). If you'd rather try and isolate via plastic adherence/culture methods you could try one of the Serum-Free MSC specialty media which may enable you to expand MSCs without other cell populations contaminating the culture. I worked for StemCell Technologies for a long time and actually developed their serum free MSC media, so am partial to that haha.
Mesencult XF should grow the MSC out (I was the lead developer on that media) but there's still a chance some of the colonic epithelial cells will grow.. Plate at low densities to try and encourage the clonogenic growth of CFU-F and hopefully hinder the growth of epithelial cells. To confirm that what you end up with are MSC, you'd need to do the standard differentiation assays- chondro, osteo and adipo, and maybe run some FACS panels to look at phenotypes.Good luck!
No. This is for whole digest- If your protocol has a step to denude epithelial cells prior to culture that will obviously help. Without having seen your processes it is hard to take such steps into account, but in most of these protocols nothing is absolute and some epithelial cells will likely make it thorough. What-s your protocol- a digest to split the epithelial upper from the underlying muscularis targeting the submucosal junctions?
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