I am now trying to proliferate early passages of hematopoietic stem cells from UCB and using IL-3, GM-CSF and SCF supplemented StemPro media. Approximate doubling time is 5 days slowly. I want to induce proliferation to avoid dormancy level of HSCs. Any suggestions??
Dear Kamer,
Check out Guy Sauvageau's work from IRIC in Montreal. He identified compounds that specifically expand HSCs from cord blood. I have attached a paper, but I'm sure there's more information in his more recent work.
http://science.sciencemag.org/content/345/6203/1509.full
All the best,
Michael
Put them on feeder layer of MS-5 or HS-5 or MSC ...etc
P.S. I don't think you will get dormancy anyway in any case with and without feeder. When they don't proliferate they just loose the CD34 expression and differentiate. Dormancy has a different concept.
Dear Hasan,
I am using feeder free media and I tried with and without GM-CSF, without it, they just stopped proliferating and started to differentiated but after adding GM-CSF, I saw proliferation again. That's why I thought they were in a dormant state. Because some articles showed that hematopoietic stem cells (not precursors) can alter the dormancy by themselves (I dont know how yet). But I will check for the CD34 expression thanks for the advice.
Bests,
Kamer
Differentiated cells might proliferate for a while before they stop when they reach the final step of differentiation. GM-CSF would help to keep them happy but it wont impact on CD34 expression. I would suggest using a feeder cells as the steaminess is not only maintained by the media cytokins but also with the cell-cell contact which the feeder cells provide. I will also strongly suggest using antagonist of aryl hydrocarbon receptor (AHR) to maintain CD34 expression high. STEMCELL has a compound called (SR1) which you may consider. Good luck
Hi Kamer Burak Isci as Michael Förster suggested Dr. Guy Sauvageau has identified the small molecules UM171 and UM729 which can be used to expand cord blood-derived CD34+ cells efficiently in vitro.
We have developed an optimized protocol using serum-free StemSpan SFEM II medium, CD34+ cytokine cocktail and UM171 and see 80X - 100X expansion of CD34+ cells (10X expansion of more primitive CD34+CD45RA-CD90+ cells) after 7 days of culture.
Check it out here if you are still in need of some guidance for your experiments:
https://www.stemcell.com/technical-resources/educational-materials/tech-tips/serum-free-expansion-of-cd34-cells-with-sfem-ii.html?utm_source=researchgate
Let me know if you have any questions, good luck!
Amanda
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