I have been culturing LSK cells isolated from C57BL/6J mice (ages ~8-12 weeks) after sorting using base stem cell media (with 10% FBS, 2-ME, MβCD) + SCF (5-25ng/ml). I am trying to look at proliferation with a modified WNT system. 7-10 days later when I go to analyze my cells with FACS, I do see expansion but 65-80% have differentiated (or at least, lost Sca and ckit positivity) with SCF alone which seems more than what I would expect based on my literature review. Plus, many of the cells are adherent which I have been told would likely be contaminating cells (e.g. fibroblasts). The protocols that I have read on stem cell culturing do say that you can use trypsin or accutase for the adherent cells but is that not routinely done? This may be a very a basic problem that I am not understanding.
SCF alone is insufficient to maintain the progenitors. Almost a default differentiation mode of these cells (when unhappy) is towards macrophages...which are adherent and likely. Hard to recommend media as long as one does't know what your aim is. Maybe the Lodish cocktail (Scf+Thpo+IGFBP2+Fgf1+Angptl1 or the older one Scf+Thpo+Igf2+Fgf1).
Although I work primarily with human CD34+ HSPC (from cord blood or mobilized peripheral blood), I agree with Dirk's answer. For the human counterparts to your cells, SCF alone is definitely not enough to maintain them in a naive, stem or early progenitor state. There are quite a few papers evaluating culture conditions for expanding HSCs though ( if that is your aim ). Otherwise, please specify the lineage that you are ultimately trying to differentiate towards...?
Thank you all for your responses. Others in my lab work with macrophages so I considered that the adherent cells may be macrophages. However, if I do not add SCF, the cells start to die within a few days so that made me question if the adherent cells were macrophages or even fibroblasts. I also considered mast cells (although I cannot find convincing evidence of their plate adherent properties).
As for the SCF, it is clear that it alone will not maintain murine HSCs in a naive state and I am counting on there being differentiation with SCF alone since it is my control. However, I expected not to lose nearly all of my ckit and sca positivity within 1 week given that others have said that SCF by itself, should maintain short-term repopulating cells for up to 10-14 days in vitro (Li and Johnson, Blood 1994). I think that my media composition is contributing to my dilemma so I will focus on that now.
Nahal, I agree with Dirk that the differentiated cells should be macrophages, as the the macrophages are tightly adhesive to the dish. according to my experiments, culturing with SCF alone can maintain LSKs for at least 11 days. May be your should use SCF at higher level, 50 ng/ml should be better. Also, using IMDM medium, insulin, transferrin will help maintaining the LSKs.
Thank you, Xufeng. Can you please send me the culture media recipe you are using or the reference of the publication it comes from?