Help from any friend working with BMSC- Suggestion needed - BMSC isolation & differentiation (Feb/20/2007 )

Hi friends,

i am new to stem cell research. My boss has decided to start stem cell work in collaboration with a orthopaedic surgeon. i collected few details from net regarding isolation of Bone marrow stem cells. briefly what i did was, i isolated mononuclear cells by Ficoll density gradient centrifugation from Bone marrow aspirate. buffy coat which i got was then suspended in RPMI-1640+FCS. cell no.was very good. but now the question comes that how to identify stem cell from population of MNCs i got?after 24 hrs i replaced the medium with a fresh . the protocol i got from a research paper.
Next i did was that after 24 hrs i added dexamethasone 10-8M and incubated for another few days. i kept on observing the growth and found that morphology of cells were now changed from round to spindle shape and then they appeared like fibroblast cells and after 10 days the whole surface of T-25 flask was covered with fibroblast like cells stroigly adherent to base.
Now the problem was that how should i make myself sure that the cells are osteoblast or what else? i ve found few reference mentioning that dexamethasone leads to differentiation of BMSC to osteoblasts. Is this true?
My big question is : Is there any parameter available which i can use as a marker for stem cells, so that i can make myself sure that i m also getting stem cells in my MNCs population which i isolate by ficoll seperation?what are markers for osteoblast. actually we dont have FACS and flouroscence facility. so i m in need of simple markers.
Please help me out in this regard. i found this forum a best guide thru which i can learn the things. My sir doesnt know anythin abt stem cell research and i ll ve to take help of you all who are working in this feild. i am very much interesting in this feild and want to pursue my career in stem cell research.
Thanks a million. hope to hear something from my friends.
Regards

Hi,
First of all, I assume that you are trying to get MSC from Human bone marrow, right? so this is only applicable for human MSC. other species has different marker or methods so...
anyway,
we do MSC separation quite often in our lab, and we normally use magnetic separation (from miltenyi biotec?) with STRO-1 antibody. So, we dilute BM with IMDM, and do Ficol, then go to magnetic separation.
in this methods, we have pritty good purity of MSC (we usually characterize seperated cell with FACS to make sure).
I understand that you dont have FACS, so it might be very difficult for you to make sure that you got MSC or not.
but that's what we do, so just as one of suggestion...

Good luck

Thanks for your suggestion Rnotk.
ll contact you if i ll need any help for MSC