Fwd: Re: problems with CD34 cells

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Dear All,

It is of course, not easy to grow CD34+ cells whilst preserving their 
stemness - many people have tried and unfortunately failed miserably 
despite extensive efforts.
However, I have found the work from Dr Wanda Piacibello's group in Torino 
very useful regarding this issue, and I advise you looking up some of her 
publications on PubMed, (eg. Ex vivo expansion of human adult stem cells 
capable of primary and secondary hemopoietic reconstitution, Gammaitoni et 
al, Experimental Hematology 31 (2003) 261–270).
  Earlier this year I spent a week in her lab playing with CD34+ cells, 
MACS and picking up her techniques etc, then returned to our lab and 
replicated the experiments.  To give a brief summary, I found two rounds of 
MACS would yield me with CD34+ cells of greater than 98% positive.  They 
were seeded under her conditions at a density of 20,000 cells per well 
(1ml, with SCF, IL6, TPO and Flt3 ligand), and rapidly took off after a 
couple of days lag.  I found that after 2 weeks of culture I still had 38% 
of my cells in culture were CD34+, and I had a heck of a lot  more cells 
than I started with.  By setting up several wells in parallel, then pooling 
them, and re-MACing them, you can maintain a continuing culture of CD34+ 
cells and get expansion!  I havent done any transplantation studies with 
them, but her group has reported that they are capable of reconstituting 
the haemopoietic system well.
Obviously, its not an easy process , but it can be done, and my own 
experiments have confirmed this.

I hope this may be useful to you and anyone else working in this field.

Best of luck!

Regards,

Dr Mike Orford,
The Cyprus Institute of Neurology & Genetics,
Nicosia,
Cyprus

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