From: Douglas Dooley (redxr@teleport.com)
Date: Thu Sep 24 1998 - 13:02:29 EST
Dear Flowers --
I would like to commend Rob Sutherland for his learned discussion of
CD34+ subsets, with particular emphasis on CD38- cells. We, too, have
found that isotype controls have been less than stellar in defining the
cut-off for the CD38+ subfraction within CD34+ cells. Frequently, the
(negative) peak of the isotype control for CD38 may be shifted somewhat to
the right compared to the negative peak of cells stained with CD38.
It has been suggested that CD34+CD38- cells in mobilized blood are too
rare to obtain for functional or phenotypic analysis. On this point we
disagree. Mobilized 34+38- cells can be obtained quite easily provided
sorting is preceded by a CD34+ cell enrichment step e.g., Miltenyi magnetic
beads. When the pre-enrichment step is performed (one column pass) the
starting population for sorting averages 60-80% CD34+ and the CD38- subset
is readily seen. You will find, however, that the proportion of CD34+
cells which is CD38- varies ~ 10-fold between patients (perhaps 1-10%). It
may also be affected by the time at which the mobilized blood was
collected, since some believe that more primitive cells may come into
circulation earlier than bulk CD34+ cells. (Comments appreciated here).
Finally, with regard to the utility of the CD38- marker on mobilized
CD34+ cells, we have found consistently over 4-6 years that this fraction
is markedly different from CD34+CD38+ cells. For example, compared to
CD34+ CD38+ cells, the CD34+CD38- fraction from blood (1) has greater
proliferative potential, (2) is more highly enriched for LTCIC, (3) is much
more responsive to Flt3 ligand, (4) expresses Flt3 gene at higher levels
and (5) is more sensitive to TGF-beta inhibition.
However, we wish to point out a major caveat: the significance of the
CD34+CD38- phenotype may be totally different when observed in stem cell
expansion cultures. That is, when cultures are initiated with CD34+CD38-
cells and examined later, an increase in the number of CD34+CD38- does not
necessarily mean that there has been renewal or expansion of primitive
cells. This is because the CD34+CD38- cells recovered from the culture are
a mixture of high proliferative quiescent cells which never cycled and low
proliferative cells which did cycle. Thus, the phenotype alone can be very
misleading when used to analyze expansion cultures.
Hope this helps.
Doug Dooley
Barbara Oppenlander
Stem Cell Lab
American Red Cross
Portland, OR
redxr@teleport.com
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