Does anyone have experience on differentiation of mouse induced pluripotent stem cells (iPSCs) into hematopoietic fate? I need an experimental design to differentiate my iPSCs into hematopoietic lineage but not a specific lineage. I need to know which culture supplements are the minimum requirements and finally which markers and functional tests should be analyzed to confirm the successful differentiation (which genes and surface markers must be analyzed by qPCR and flowcytometery?). owing to the limited grant, I appreciate the answers with the minimum compulsary markers. thank you.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706875/
This review is one of the first I have hit on Pubmed, that would be a good starting point, with plenty of practical examples. My experience is not with mouse cells, but I think the less expensive system is a co-culture with OP9: you need very few cytokines and no fancy medium, just fetal bovine serum and IMDM. You will find plenty of protocols for hematopoietic differentiation with OP9!
Good luck!
I would look at functional testing. It's more both more definitive and cheaper. Less likely to tell you what you want to hear (consider that an advantage or a disadvantage, depending on where those grants come from?).
Colony-forming assays. Chemotaxis. Clotting. Hemoglobin / pseudoperoxidase staining. Endocytosis. Free radical production. If you have those, you wouldn't technically need flow, unless you want to publish a paper, in which case your reviewer will want it, for no reason that particularly convinces me.
It's easy to get a cell with all the right flow markers and no function. It's impossible to get a cell that functions right and doesn't have the right flow markers. (If it was possible, it might still be valuable both as basic science result and cell therapy candidate, but I digress ;-)
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