Is there any review about hematopoieitic differentiation of mouse embryonic stem cells on OP9 cells with complete profile of markers expressed? I have tried in all possible ways to find the markers expressed at each stage of differentiation, but I could not. I am supposed to do flow analysis and I am stuck with no information about the markers to be analyzed except CD45 and Flk-1.
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Hi there,
The Medvinsky group at the University of Edinburgh use this system for their murine haematopoetic work all the time. Check out their lab page and publication page for many relevant references!
http://www.crm.ed.ac.uk/research/group/ontogeny-haematopoietic-stem-cells
Hello
See this links may help you .
Good Luck
http://stemcellassays.com/2011/06/hematopoietic-differentiation-of-mouse-es-cells-using-op-9-co-culture-system-video-protocol/
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058123
http://www.jlr.org/content/47/2/450.full
OP9 are particularly difficult to maintain and although they are useful for ES cell differentiation into blood cells, there are alternatives. In my lab we are routinely differentiating ES cell into blood cells efficiently without OP9 cells. We get the same populations as the one you'll find with OP9 based ESC differentiation. In that context, I would suggest to use antibodies against ckit, CD41, CD31 and CD45 if you wish to detect blood cells. For endothelial cells, I suggest an antibody against VE-cadherin and Flk1. As you may know the anti-Flk1 antibody can also be used to detect mesodermal progenitors giving rise to blood, endothelial and vascular smooth muscle cells. Let me know if you need clarification.
Saleh Alkarim: Thank you for the links provided.
Christophe Lancrin:
first of all, I am excited with happiness to see a suggestion from you as I am currently reading your Nature 2009 published data on hemogenic endothelium. I am aware of a few methods of OP9-independent hematopoietic differentiation, but, I found the OP9 cost-effective and relatively easy in terms of labor (please correct if I am wrong). But I found that in most of the reported work in ES/OP9 co-culture, cytokines were added to enhance differentiation; whereas I am not adding them, as a result, the timeline for emergence of differentiated cells might be different (I suppose). OP9 method takes long time for each experiment (14-15 days), with no external addition of cytokines it may take more time which I am not sure about.
Dear Wulligundam,
I'm glad you find my paper of interest.
Going back to your OP9/ES experiment, why are you not adding cytokines? Is it your goal to find out what happens when you don't use growth factors? Have you tried the experiment with cytokines and compared it with the literature?
Christophe
Dear Christophe,
I am currently doing a preliminary study and due to some reasons I cannot use cytokines. I expected a decrease in the number of differentiating cells, detectable by flow cytometry, but, I am realizing that the time-line of differentiation itself changes in the current cytokine-independent method. Based on your suggestion, I have included the VE-Cadherin and CD41 also in the list of markers to be analysed. In the current situation, firstly, I would like to define the time-line of differentiation and then proceed for flow analysis. Your suggestions are most welcome.
I thank you all, for your valuable inputs. I would like to ask a question about culturing the OP9 and mES/OP9 cells. In literature, there is a difference in composition of differentiation medium used. Some have used with beta-mercaptoethanol (beta-ME) where as some did not. As Beta-ME is a know radical scavenger, I am currently using in my experiments. I would like to know if there is any role of beta-ME in differentiation?
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