Full text of DOI:10.1038/sj.leu.2402254

Leukemia (2001) 15, 1677–1680
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SPOTLIGHT ON HEMATOPOIETIC STEM CELLS: LOOKING BEYOND DOGMA
Introduction
JA Nolta and CT Jordan
Children’s Hospital of Los Angeles, Los Angeles, CA, USA
Some of the long-established dogma concerning normal hem-
atopoietic and leukemic stem cell phenotype and function
have been questioned in the past few years. As a result,recent
publications have been exciting and presentations at Hematol-
ogy meetings have sometimes been highly controversial. This
section will address some of the new cutting-edge experimen-
tation that is going on in the field,with special attention to
novel,well-documented evidence that alters our perception
of old stem cell dogma.
depleted human stem cell populations in their experiments,
until further knowledge is gained that will allow more
accurate isolation.
An alternative method to isolate primitive stem cells that is
rapidly gaining popularity,is based upon the enhanced
capacity of the most primitive stem cell populations to efflux
Hoechst dyes,as has been described by the groups headed
7–9
by M Goodell and R Mulligan. This strategy is based more
on function than on phenotype. Dr Goodell will write a
review for the ‘Beyond dogma’ section on the Hoechst dye-
excluding ‘side population’,or ‘SP’ cells that her laboratory
has characterized. Similarly,Dr Brian Sorrentino’s laboratory
has determined that the Bcrp1/ABCG2 molecule is responsible
for effluxing the Hoechst dyes from murine and human hema-
topoietic cells,and may be a novel hematopoietic stem cell
marker. Of interest,Hoechst dye-excluding SP populations
can be detected in multiple tissues,including muscle,brain,
liver and kidney,in addition to bone marrow. This population
is thus important in studies of so-called ‘stem cell plasticity’
discussed briefly below.
An initial topic that has been recently reviewed in this area
is that evidence now exists to support the presence of both
murine and human hematopoietic stem cells that lack
expression of the CD34 marker (reviewed in Refs 1–3). There
appears to be a degree of reversibility of expression of CD34
4
at the stem cell surface,in both murine and human stem
5
cells. The appearance and disappearance of the cell surface
+
protein could complicate CD34 cell immunoisolation
methods that are designed to debulk or T cell deplete bone
marrow or peripheral blood products for transplantation.
+
However,the reversibility data suggests that the CD34 stem
−
cell pool may be able to generate the CD34 pool,and vice
Rodent transplantation studies have suggested the existence
of totipotent stem cells in the bone marrow compartment,cap-
able of generating not only blood cells,but also liver or mus-
versa. Whether the two pools have different generative poten-
tials,either in the number or in the type of progeny that they
produce,remains to be determined.
10
cle. Lagasse et al, in Markus Grompe’s group reported that
+
−
+
One of the initial reviews for the ‘beyond dogma’ section,
in this issue,is by Dr Mick Bhatia’s laboratory and suggests
that an earlier stem cell marker than CD34 may be the cell
surface molecule CD133,recognized by the AC133 antibody
produced by Miltenyi Biotec (Auburn,CA,USA). Dr Bhatia’s
c-kit Thy-1.1 (lo) Lin sca-1 stem cells,isolated from the
bone marrow of adult mice,rescued the liver defect in the
FAH− mouse,an animal model of tyrosinemia type I,by
restoring biochemical function to the liver. The same popu-
lation of cells was also capable of radioprotection of the leth-
/−
+
−
−
10
+
laboratory has demonstrated that the AC133 CD34 lin
ally irradiated mice,in the same study.
The c-kit Thy-1.1
−
+
population isolated from human mobilized peripheral blood
(lo) Lin sca-1 (KTLS) population of murine stem cells had
+
11–13
can generate CD34 progenitors. These data are discussed in
been shown to be radioprotective,
but the elegant demon-
his review in this issue. Further reviews and primary data on
the phenotype and function of primitive stem cells,and on the
regulation of expression of CD34 and other stem cell surface
markers will be solicited for the ‘Beyond Dogma’ section.
Other data to be presented in this Spotlight section will con-
cern the reversibility of not only CD34 but also of CD38 and
HLA-DR,molecules that have been used as a basis for iso-
lation procedures,and that are now found to change in cul-
ture without apparent alterations in function to accompany
the change in phenotype. These data make the reisolation of
cultured stem cells,such as those defined by the
CD34 /CD38 phenotype,highly unreliable. The dissociation
of phenotype and function,as Dr John Dick has described,is
something to be carefully considered with any population of
stem cells that has been identified primarily by cell surface
markers. Many laboratories are now simply using lineage-
stration by Lagasse et al that the same population could also
generate functional hepatocytes was highly novel. There may
have been one single cell type that was responsible,a primi-
tive totipotent cell,or there may be two separate cells with
liver and blood regenerating capacity within the stem cell
population that was used. The different theories of plasticity
are shown in Figure 1.
14
Jackson et al demonstrated that a portion of the Hoechst
dye-excluding SP cells isolated from the skeletal muscle of
adult mice were capable of hematopoietic differentiation. In
another study,murine SP cells isolated from bone marrow
were found to assimilate into the muscle of a dystrophin-
+
−
6
15
deficient mouse after systemic infusion. In a third mouse
+
−
model of stem cell plasticity,c-kit /lineage cells isolated from
rodent bone marrow had the capacity to heal cardiac
16
infarcts. These data demonstrate that cells capable of gener-
ating blood can be found in unexpected places,and that mar-
row-derived cells can display potentials that were not pre-
viously thought to exist in the marrow compartment. These
revolutionary studies therefore challenge the old dogma that
bone marrow stem cells exist only to make blood.
Correspondence: JA Nolta,Childrens Hospital of Los Angeles,4650
Sunset Blvd,Mailstop 62,Los Angeles,CA 90027,USA; Fax: 323
6
60 8736
Received 23 April 2001; accepted 19 June 2001
There may be uncommitted,totipotent cells within the bone

Spotlight on hematopoietic stem cells
JA Nolta and CT Jordan
1678
due to a lack,so far,of appropriate in vivo models for studying
human stem cells that may have the capacity to generate mul-
tiple tissues. In retrospective studies,Neil Theise,Diane
Krause and colleagues demonstrated that in humans,hepato-
cytes and cholangiocytes could be derived from bone mar-
18
row. They analyzed archival liver specimens from female
recipients of bone marrow transplantations from male donors,
and found Y chromosome-positive hepatocytes and cholangi-
18
ocytes in the female BMT recipients. This observation sug-
gests that marrow-derived stem cells can generate liver in
humans. Drs Theise and Krause will provide a review of the
very exciting work from their laboratories in the ‘Beyond dog-
ma’ forum.
The possibility that a population of totipotent,uncommitted
stem cells may exist in the bone marrow is intriguing,and if
a human counterpart exists,it could have immense clinical
benefit. We can imagine that in the future a marrow-derived
stem cell transplant could be used to repair injury to a
patient’s liver or heart,rather than the patient needing to
endure the long wait for a cadaveric donor. Totipotent,pre-
hematopoietic stem cell populations may also be identified,
in the future,that are free of leukemic cells,since most of the
stem cell phenotypes identified to date can contain both nor-
mal and leukemic cells,as discussed below.
Figure 1
Stem cell theories: Left panel: Two stem cells with differ-
ent fates reside in the same location and are isolated together,giving
rise to two tissues in subsequent assays. Central panel: Stem cells
restricted to one fate can trans-differentiate to a different tissue,depen-
dent on the microenvironment or other cues. Right panel: One totipot-
ent,or uncommitted stem cell can generate two different tissues.
marrow that can generate both blood and other tissues such
as muscle or liver. An alternative explanation for the observed
Another important aspect of stem cell biology that will be
addressed in the ‘Beyond dogma’ section concerns the origins
of hematologic malignancy. Recent studies have charac-
terized stem cell populations for both acute and chronic myel-
ogenous leukemia and have greatly enhanced our understand-
ing of the biology of these diseases. For example,stem cells
for acute myelogenous leukemia (AML) have now been
phenotypically described in a fashion similar to that of normal
stem cells. Studies by Dick and colleagues have shown that
‘
plasticity’ of marrow-derived stem cells could be that liver or
muscle-committed cells reside in the bone marrow,in
addition to the blood-forming cells (Figure 1). To discriminate
between the ‘totipotent stem cell’ and ‘two stem cell’ theories,
retroviral or lentiviral marking of the individual stem cells will
need to be done,with tracking of the same stem cell into pro-
geny of two disparate cell types using clonal integration analy-
sis (Figure 2),as our group has previously described in the
transduction of human hematopoietic stem cells that were
primitive enough to generate both T lymphoid and myeloid
+
−
primary AML cells with a CD34 /CD38 phenotype are suf-
ficient to perpetuate leukemic growth in the xenogeneic
19,20
NOD/SCID mouse model system.
Importantly,stem cell
17
progeny.
engraftment in this model is dose-dependent and sufficient to
There are few data indicating that human counterparts may
exist for the murine and rat stem cells that display plasticity,
transplant secondary recipients. Similarly,a report by Blair et
al has shown that CD34 /CD71 /HLA-DR AML cells can
21
+
−
−
engraft NOD/SCID mice and maintain long-term culture
22,23
growth in vitro. Of interest,Blair and colleagues
have also
shown that AML stem cells lack expression of Thy-1 and
CD117. These observations are important because they rep-
resent the first reports of antigenic features of primitive leuke-
mia cells that differ from normal stem cells. More recently,
further studies have shown that CD123 is differentially
expressed between malignant and normal stem cell popu-
lations,with high levels of CD123 found on leukemic stem
24
cells. New reports of markers that could potentially be used
to separate leukemic from normal stem cells are sought for
the ‘Beyond dogma’ section.
The availability of antigenic markers that distinguish leu-
kemic from normal stem cells has permitted more detailed
molecular analyses of primitive leukemic cells. For example,
two recent studies have described aberrant expression of
tumor suppressor genes and the transcription factor NF-kB in
25,26
primitive AML but not normal hematopoietic cells.
Several
interesting advances have also been made for chronic myelo-
genous leukemia (CML),which will be reviewed in the ‘New
dogma’ section by Connie Eaves and her colleagues. Perhaps
most importantly,it has been shown that primitive CML cells
appear to possess autocrine cytokine activity that may be cen-
Figure 2
Clonal integration analysis to track tissue origin. One
definitive way to determine that cells from two disparate tissues are
derived from a common stem cell is through the use of clonal inte-
gration marking. Retroviral vectors integrate randomly into the cellular
DNA of the target stem cell,providing a clonotypic marker that can
be identified and tracked in the progeny of the stem cells by sensitive
PCR-based methods.
27–29
tral to their cell cycle activation and differentiation.
In
addition,it has also been shown that at least some CML stem
cells are highly quiescent,much like their normal stem cell
Leukemia

Spotlight on hematopoietic stem cells
JA Nolta and CT Jordan
1
679
3
0
counterparts. Taken together,these studies,as well as others,
are shedding new light on the nature of leukemogenesis and
the properties of primitive malignant cells.
lation and functional properties of murine hematopoietic stem
cells that are replicating in vivo. JExp Med 1996; 183: 1797–1806.
8
Goodell MA,Rosenzweig M,Kim H,Marks DF,DeMaria M,Par-
adis G,Grupp SA,Sieff CA,Mulligan RC,Johnson RP. Dye efflux
studies suggest that hematopoietic stem cells expressing low or
undetectable levels of CD34 antigen exist in multiple species. Nat
Med 1997; 3: 1337–1345.
9 Storms RW,Goodell MA,Fisher A,Mulligan RC,Smith C. Hoechst
dye efflux reveals a novel CD7(+)CD34(−) lymphoid progenitor in
human umbilical cord blood. Blood 2000; 96: 2125–2133.
0 Lagasse E,Connors H,Al-Dhalimy M,Reitsma M,Dohse M,
Osborne L,Wang X,Finegold M,Weissman IL,Grompe M. Pur-
ified hematopoietic stem cells can differentiate into hepatocytes
in vivo. Nat Med 2000; 6: 1229–1234.
11 Bowers E,Tamaki S,Coward A,Kaneshima H,Chao CC. Differing
functional recovery of donor-derived immune cells after purified
haploidentical and fully mismatched hematopoietic stem cell
transplantation in mice. Exp Hematol 2000; 28: 1481–1489.
Exciting data have also been recently reported in the areas
of stem cell homing after transplantation. The use of immune-
deficient mouse xenograft systems to study human stem cell
homing has revolutionized the field,and molecules that acti-
vate homing receptors have been identified. Dr Tsvee Lapidot
will contribute a mini-review on the interesting work done
in his laboratory on SDF-1 activation of the CXCR4 homing
receptor,thought in previous years to be most important on T
cells as the co-receptor for the HIV virus. Although somewhat
1
3
1
controversial, the work of Dr Lapidot and others has shown
that the CXCR4 receptor plays an additional role in the in vivo
homing and engraftment of stem cells.
Another exciting area of research in the stem cell homing
area is discussed in this issue by Ed Srour,who along with
Peter Quesenberry and Pamela Becker’s groups,have shown
that non-cycling hematopoietic stem cells home back to the
32,33
1
2 Uchida N,Friera AM,He D,Reitsma MJ,Tsukamoto AS,Weiss-
man IL. Hydroxyurea can be used to increase mouse (−kit+Thy-
1
. 1(lo)Lin-/loSca-1(+) hematopoietic cell number and frequency
in cell cycle in vivo. Blood 1997; 90: 4354–4362.
34,35
bone marrow more efficiently than cycling cells.
This
13 Uchida N,Tsukamoto A,He D,Friera AM,Scollay R,Weissman
IL. High doses of purified stem cells cause early hematopoietic
recovery in syngeneic and allogeneic hosts. JClin Invest 1998;
research is highly important for the field of ex vivo stem cell
expansion and for the field of gene therapy,which primarily
concentrates on inducing cells to cycle to allow integration of
mitosis-dependent viral vectors. It is possible that the success
of gene therapy could be significantly improved if the stem
cells could be returned to the quiescent phase after viral entry
and integration but prior to transplantation. Thus,although
controversy exists at the moment,the issues of stem cell cycle
and homing are highly important. Dr Ed Srour will discuss the
controversies that currently exist in the area of hematopoietic
stem cell homing.
In addition to the exciting reviews that we have planned for
this section,we are soliciting suggestions for other reviews or
primary data that fit with or augment the themes described
above. For participation in the ‘Spotlight on hematopoiesis:
looking beyond dogma’ section,please send ideas for reviews
to jnoltaȰchla.usc.edu or submit completed manuscripts to
the Leukemia Editorial Office in Paris labeled: ‘Spotlight on
Hematopoiesis (Beyond dogma section) for the attention of Dr
JA Nolta (normal stem cells) or Dr C Jordan (leukemic stem
cells)’.
101: 961–966.
1
4 Jackson KA,Mi T,Goodell MA. Hematopoietic potential of stem
cells isolated from murine skeletal muscle. Proc Natl Acad Sci
USA 1999; 96: 14482–14486.
5 Gussoni E,Soneoka Y,Strickland CD,Buzney EA,Khan MK,Flint
AF,Kunkel LM,Mulligan RC. Dystrophin expression in the mdx
mouse restored by stem cell transplantation. Nature 1999; 401:
390–394.
6 Orlic D,Kajstura J,Chimenti S,Jakoniak I,Anderson SM,Li B,
Pickel J,McKay R,Nadal-Ginard B,Bodine DM,Leri A,Anversa
P. Bone marrow cells regenerate infarcted myocardium. Nature
1
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7 Nolta JA,Dao MA,Wells S,Smogorzewska EM,Kohn DB. Trans-
duction of pluripotent human hematopoietic stem cells demon-
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mice. Proc Natl Acad Sci USA 1996; 93: 2414–2419.
8 Theise ND,Nimmakayalu M,Gardner R,Illei PB,Morgan G,
Teperman L,Henegariu O,Krause DS. Liver from bone marrow
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9 Lapidot T,Sirard C,Vormoor J,Murdoch B,Hoang T,Caceres-
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