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Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2545-2547
INTRODUCTION: FOCUS ON HEMATOLOGY
CD34+ or CD34 : Does it Really Matter?
By
Margaret A. Goodell
From the Center for Cell and Gene Therapy, Baylor College of
Medicine, Houston, TX.
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ARTICLE |
THE CELL SURFACE glycoprotein CD34 has
been the focus of intense interest ever since its discovery on a small
fraction of human bone marrow cells.1 The CD34+
population appears responsible for most, if not all, of the
hematopoietic activity in bone marrow, including colony formation in
short-term assays,1 the maintenance of long-term in vitro
cultures,2 and the expansion and differentiation of blood
cell subsets in immunocompromised mice.3 Successful
engraftment of baboons with bone marrow highly enriched for
CD34+ cells4 led to the widespread use of
CD34-enriched populations in human transplantation. In general,
patients transplanted with marrow or mobilized peripheral blood
enriched for CD34+ cells engraft rapidly and seem to have
fewer transplant-related complications.5-8 Devices for the
enrichment of CD34+ cells have been a commercial success
and the object of infamous corporate battles.
However, niggling worries about the phenotype of the most primitive
hematopoietic stem cells began to surface about 3 years ago and now
challenge the preeminence of CD34 as a stem cell marker. Experiments on
bone marrow from both mice and humans suggested that at least some
primitive progenitors lacked CD34 and were capable of generating
CD34+ stem cells.9-12 This new concept stirred
enormous controversy among investigators who had taken CD34 from the
bench to the bedside, guided by ostensibly sound scientific principles.
It challenged not only the interpretation of a wealth of experimental
data on the potency of CD34+ cells (and the lack, thereof,
of CD34 cells), but also the wisdom of applying them
in clinical practice. The tacit suggestion was that researchers and
clinicians alike may have been unwittingly discarding the very best
stem cells.
After overcoming their initial surprise, hematologists worldwide
undertook to address the critical questions surrounding this novel
hypothesis. In this issue of BLOOD, Sato, Laver, and
Ogawa13 present an elegant study on the
expression of CD34 on long-term reconstituting stem cells in the mouse.
Their report was a pleasure to read due to the simplicity of the
experiments, the strength of the conclusions, and the clarity of the prose.
In the mouse, one has the luxury of testing the activity of
hematopoietic stem cells in the most rigorous way: by transplanting test populations into myeloablated recipients and examining peripheral blood for the progeny of test cells over the course of many months. Because of the plethora of inbred mouse strains, cell populations differing at only 1 or 2 genetic loci can be readily transferred from
one strain to another, thereby reducing the number of potentially confounding variables. The marker du jour is CD45 (still often called
by its older murine nomenclature, Ly-5), of which 3 alleles exist that
differ by a few amino acids, each being recognized by specific
monoclonal antibodies.14 Because CD45 is expressed on all
nucleated peripheral blood cells, it can be used to distinguish progeny
of test populations by flow cytometry analysis for the CD45 allele in
conjunction with lineage markers.
Sato et al13 used this powerful system to revisit the
question of whether CD34 is expressed on primitive murine hematopoietic stem cells, as defined exclusively by bone marrow transplantation. They
purified CD34+ and CD34 cells from
Ly-5.2 mice and transplanted these subsets into sublethally irradiated
Ly-5.1 recipients. They also administered to the recipients a small
dose of compromised Ly-5.1 marrow (bone marrow depleted of stem cell
activity by serial transplantation), providing short-term peripheral
blood cell production and perhaps other cells and factors that may
support the engraftment of sorted cells.
When stem cells from normal mice were sorted, the investigators
observed clear segregation of stem cell activity into the CD34 population. At 2 and 5 months after
transplantation, all mice transplanted with small numbers of
CD34 cells showed high numbers of Ly-5.2 progeny.
Mice transplanted with 5 times as many CD34+ stem exhibited
little to no repopulation from the sorted cells. The results were
dramatically different when the investigators used bone marrow from
mice that were treated 2 days earlier with 5-fluoruracil (5-FU). Toxic
to dividing cells, 5-FU rapidly depletes the marrow of committed
progenitors and appears to recruit dormant hematopoietic stem cells
into cycle.15 When the investigators sorted stem cells from
these animals, using a strategy identical to that applied to normal
marrow, they found the stem cell activity to be fairly evenly
distributed between the CD34 and CD34+
compartments, regardless of whether the recipients were examined at
short (2-month) or long (8-month) intervals after transplantation. These results suggested that CD34 on quiescent stem cells might be
upregulated in response to proliferation signals and, as such, may be a
marker of activated stem cells. In pursuit of this idea, Sato et
al13 stimulated normal CD34 stem cells
in vitro with stem cell factor and interleukin-11. After 1 week of
culture, they observed a 1,000-fold expansion of the
CD34 population and the acquisition of a
CD34+ phenotype by 75% of the cells. When the
CD34+ cells and CD34 cells were sorted
from these cultures and transplanted into mice, only the latter gave
rise to differentiated progeny. These results showed that
CD34 stem cells can generate CD34+ cells
and that at least some of the new CD34+ cells are long-term
reconstituting stem cells, because they were associated with high level
engraftment up to 5 months after transplantation. The remaining
cultured CD34 cells had no apparent stem cell
activity in vivo.
Finally, the investigators asked whether CD34+ stem cells
could revert to a CD34 phenotype. Taking bone marrow
from mice that had been transplanted with CD34+ stem cells
derived from 5-FU-treated donors, they sorted CD34+ and
CD34 stem cells and transplanted them into secondary
recipients. At 2 and 5 months posttransplantation, they found that only
the CD34 stem cells had generated progeny. Thus,
some of the stem cells originally expressing CD34 in the 5-FU-treated
donor marrow lost the marker after transplantation.
In summary, as diagrammed in Fig 1A,
Ogawa's group has shown that a population of primitive, transplantable
hematopoietic stem cells starts out as CD34 in
normal mice. At least some of these CD34 stem cells
convert to a CD34+ phenotype upon activation by 5-FU (or
after culture), and after transplantation, the CD34+ stem
cells revert to a CD34 phenotype.
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| Fig 1.
Models of CD34 expression in bone marrow. (A) Diagram of
conclusions from the current paper by Sato et al.13 The
spheres represent arbitrary units of hematopoietic stem
cell-repopulating activity, as measured by long-term bone marrow
transplantation in mice. In normal murine bone marrow, the long-term
repopulating activity is found entirely in the CD34/low
compartment. During culture, the CD34 stem
cells expand and acquire CD34, and the CD34+ cells
contain all detectable hematopoietic repopulating activity. After 5-FU
treatment, this activity is distributed between CD34+ and
CD34 subsets of bone marrow. When bone marrow is taken
from mice previously engrafted with CD34+ bone marrow
derived from 5-FU-treated mice, the repopulating activity, measured in
secondary recipients, is restricted to the
CD34/low stem cell fraction. (B) Model of
CD34 expression on murine hematopoietic stem cells. Resting stem cells
from normal mice express little or no CD34, have a limited capacity for
self-renewal, and can convert to a CD34+ phenotype upon
activation. The CD34+ stem cells can self-renew as well
as convert to a resting CD34 phenotype. Alternatively,
they may begin to differentiate, at which point they presumably lose
their potential for self-renewal.
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These data suggest that CD34 may be a marker of activated stem cells,
but not necessarily all stem cells. This activation may represent
self-renewal or proliferation linked to differentiation (Fig 1B), an
interpretation supported by anecdotal data from other systems. When the
expression pattern of CD34 was investigated in the developing mouse
embryo, the antigen was found to be expressed on vascular endothelial
cells, particularly at the growing tips of sprouting vessels, where the
most active proliferation was occurring.16 CD34 expression
is also upregulated on rat-liver oval cells, a presumptive liver stem
cell, during hepatic regeneration.17 Nevertheless, this
CD34+ stage of stem cells is clearly not obligatory for
differentiation, because mutant mice lacking CD34 have normal
steady-state numbers of peripheral blood cells, although they have a
reduced number of hematopoietic progenitors (the hematopoietic
compartment undergoing the most extensive proliferation).18
If further experimentation bears out this intriguing link between CD34
expression and stem cell activation or self renewal, it will be
important to determine the mechanisms of CD34 regulation. Fackler et
al19 showed that surface expression of human CD34 rapidly
increased upon protein kinase C-mediated hyperphosphorylation of intracellular CD34. Thus, it will also be interesting to determine whether quiescent CD34 stem cells have intracellular
stores of CD34 message or protein that would allow such rapid activation.
This current study is the capstone on a series of studies on mouse
hematopoietic stem cells that have clearly shown that most normal
quiescent murine stem cells express very low levels of the CD34
marker.9,10,20 Some of the early contradictory
reports21,22 may be explained by the observation that
resting stem cells express low but detectable levels of CD34 as shown
when defined by antibody-independent methods.10 This
low-level of expression can cause overlap in purified subsets,
resulting in stem cell activity being measured in all populations.
What are the implications of this work for human stem cell biology? If
the human CD34 expression pattern mirrors that of the mouse, we may
expect to find substantial stores of quiescent CD34
hematopoietic stem cells tucked away in the bone marrow. Likewise, we
would expect their CD34+ counterparts to be activated with
regard to proliferation and differentiation, possibly explaining why
the hematopoietic activity of CD34+ stem cells has been
vastly easier to measure in vitro and in vivo. Recent work indicates
that human CD34 stem cells may indeed
exist.10-12 Such cells are likely to be quiescent, because
only 1 group has reported significant hematopoietic activity by them in
conventional assays,12 leading one to ask how they can be
efficiently stimulated.
We might also expect that human CD34 and
CD34+ stem cells are freely interconvertible. If their
identities do converge in vivo, perhaps we need not worry whether we
should work with CD34 or CD34+ stem
cells (as long as we are working with true stem cells: CD34 is
primarily expressed on committed progenitors). Perhaps the population
that is easiest to use and manipulate will prove to be the best choice,
at least as regards the clinic. Of course, it is still quite possible
that human CD34 expression does not entirely mimic that in the mouse in
one or more ways. The populations may not be interconvertible, or the
CD34 population may not represent an appreciable
natural reservoir for CD34+ cells. Only time, and further
investigation, will tell.
| |
FOOTNOTES |
Address reprint requests to Margaret A. Goodell, PhD,
Center for Cell and Gene Therapy, Baylor College of Medicine, One
Baylor Plaza, N1030, Houston, TX 77030; e-mail: Goodell{at}bcm.tmc.edu.
| |
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REFERENCES