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From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1992 80: 1957-1964
Phenotypic analysis of mouse hematopoietic stem cells shows a
Thy-1- negative subset
GJ Spangrude and DM Brooks
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Phenotypic Analysis of Mouse Hematopoietic Stem Cells Shows a
Thy-1-Negative Subset
By Gerald J. Spangrude and Diane M. Brooks
Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by
immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the
majority of hematopoietic stem cell activity, as defined by
long-term competitive repopulation of irradiated animals
and by a secondary transplant assay for spleen colonyforming units (CFU-S), could be localized in Ly-Gb haplotype
mice to a fraction of bone marrow cells that expresses the
Ly-GA/E (Sca-1)molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains
expressing the Thy-1.1 allele indicated that the vast majority
of activity was included in the Thy-l1OWpopulation. In contrast, hematopoietic stem cell activity found in the bone
marrow of Thy-1.2 genotype mouse strains was recovered in
both the Thy-lneg and the Thy-1IoWpopulations. However,
similar t o Thy-1.1 strains, most activity was localized t o the
Ly-GA/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between
Thy-1.1- and Thy-1.2-expressing mouse strains was not
caused by differences in the staining intensity of monoclonal
antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of
Thy-1 separated hematopoietic stem cell activity from mice
expressing the two alleles in the same manner as did
allele-specific MoAb. The results of this study show that
Thy-1 expression is not an invariant characteristic of mouse
hematopoietic stem cells, and that mice expressing the
Thy-1.1 allele are unique in that hematopoietic stem cell
activity is found exclusively in the Thy-1IoWpopulation.
This is a US government work. There arb n o restrictions on
its use.
H
systems, including the spleen colony-forming assay (CFUS), in vitro colony-forming assays, radioprotection, and
long-term repopulation.2J0 However, only a subset of ThyltoWLinnegLy-6A/E+
cells possess potent long-term repopulating potential,’1,12despite the ability of most of the cells in
the population to differentiate in widely diverse hematopoietic lineages. This separation of long-term repopulating
potential from multilineage potential underscores the difficulties in separating subsets of hematopoietic stem cells
when some degree of biologic activity is present in several
subsets.
In the present study, we have attempted to document the
presence of hematopoietic repopulating activity found within
defined subsets of mouse BM cells. While long-term hematopoietic repopulating activity was essentially restricted to
cells expressing the Ly-6A/E antigen (in Ly-sb haplotype
mice), we found heterogeneous expression of the Thy-1
antigen among long-term repopulating cells. This heterogeneity seemed to be caused by differential regulation of the
allelic variants of the Thy-1 antigen.
EMATOPOIESIS is an ongoing developmental process by which the cellular elements of the blood in an
adult organism are continually replenished. One interesting
aspect of hematopoiesis that has proven difficult to study is
the primitive hematopoietic stem cell population that
serves as the origin of all of the diverse end-stage cells
found in normal blood. These cells have proven elusive
because of their low frequency, because even within a rich
source of hematopoietic repopulating activity such as the
bone marrow they represent only a fraction of a percent of
the total cells present. In recent years much progress has
been made in designing methodologies to enrich hematopoietic stem cells from m o ~ s e l and
- ~ human5-’ hematopoietic
tissues. However, it is likely that even the most enriched
preparations of hematopoietic stem cells are not solely
composed of the most primitive cells in the hematopoietic
h i e r a r ~ h y ,which
~ ? ~ include those cells capable of sustained
production of mature progeny cells over a prolonged time
period, eg, the life span of the organism. For modern
applications of bone marrow transplantation (BMT) and
for attempts to modify the genetic contents of hematopoietic repopulating cells with the aim to correct genetic
abnormalities, it is interesting to distinguish and identify
the sources of long-term repopulating activity. Further, in
experimental systems that seek to quantitate the long-term
repopulating compartment after manipulation of the mammalian host, eg, after application of chemotherapeutic or
infectious agents, it is critical to develop reliable criteria to
identify the primitive hematopoietic stem cells.
One approach to identifying hematopoietic stem cell
populations uses monoclonal antibodies (MoAbs) to distinguish various subsets and lineages of hematopoietic cells.
These subsets can be separated and isolated by flow
cytometric methods for functional testing.2 By this approach, a population of mouse hematopoietic stem cells has
been identified by virtue of expression of the Thy-1 and
Ly-6A/E antigens in the absence of high-level expression of
a variety of antigens characteristic of differentiated lineages
of cells. This population, termed Thy-l1OwLinnegLy-6A/E+
cells, has very potent activity in most hematopoietic assay
Blood, Vol80, No 8 (October 15). 1992: pp 1957-1964
MATERIALS AND METHODS
Animals. C57BL/Ka, C57BLlKa-Thy-1.1, C57BL/lOScN,
C57BLl6-Alpha-17, C57BL/6-L~-5.1-Pep~~,
AKRIJ, and SJL/J
From the Laboratory of Persistent Viral Diseases, Rocky Mountain
Laboratories, National Institute of AllergV and Infectious Diseases,
National Institutes of Health, Hamilton, MT,
Submitted April 28,1992; accepted June 24, 1992.
The contents of this publication do not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
the mention of trade names, commercial products, or organizations
imply endorsement by the US govemment.
Address reprint requests to Gerald J. Spangrude, PhD, Rocky
Mountain Laboratories, 903 S 4th St, Hamilton, MT 59840.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
This is a US govemment work. There are no restrictions on its use.
0006-497119218008-001l$O.OOlO
1957
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1958
mice were bred and maintained in our animal facility at Rocky
Mountain Laboratories. DBA/2J mice were purchased through
Jackson Labs (Bar Harbor, ME). All animals were maintained on
acidified (pH 2.5) drinking water and autoclaved chow (Purina
Mills Inc, St Louis, MO) ad libitum.
Magnetic particle-mediated depletions. In some experiments,
BM suspensions were depleted of cells expressing the Ly-6A/E
antigen by immunomagnetic particle methodology. For control
depletions, the anti-CD5 MoAb 53-7.31313 (obtained from American Type Culture Collection [ATCC], Rockville, MD) was used
because it is isotype matched to the anti-Ly-6A/E MoAb E13
161-714(both are IgGa), labels BM cells at approximately the same
frequency (5-7% of the total number of cells), and does not react
with long-term repopulating hematopoietic stem cells. All manipulations of BM cells were performed using Hanks' Balanced Salt
Solution containing 3% fetal calf serum and 10 mmol/L HEPES
buffer, pH 7.2. The cell suspensions were incubated with a
saturating concentration of the relevant MoAb at a cell density of 5
x lo7 cells/mL for 20 minutes on ice, followed by a wash. One
milliliter of twice-washed immunomagnetic particles (Dynal Inc,
Great Neck, NY, sheep antirat Ig specificity) was added to the
washed cells and the mixture was incubated for 30 minutes at 4°C
with constant mixing. The particles with attached cells were then
removed from the solution by application of a magnetic field and
the remaining cells were collected, concentrated by centrifugation,
and the procedure was repeated. A small sample of the twicedepleted population was stained with a fluorescein-conjugated
antirat Ig reagent (Caltag Laboratories, South San Francisco, CA)
and analyzed by flow cytometry to confirm depletion of the target
population.
Hematopoietic stem cell enrichment. BM cells were prepared
from 8 to 12 young adult mice (4 to 8 weeks old) by crushing femora
and tibia with a mortar and pestle. Low-density cells were enriched
by equilibrium centrifugation on a cushion of metrizamide (Nycomed analytical grade; Accurate Chemical and Scientific Corp,
Westbury, NY) at a density of 1.085 g/mL. The low-density cells
were reacted with a saturating solution of MoAb specific for CD2
(RM-2.215), CD3 (KT3-1.1I6), CD5 (53-7.313), CD8 (53-6.7213),
Mac-1 (M1/70.15.11.5.HL17),B220, Gr-1 (RA3-6B2 and RB6-8C5,
respectively, obtained from Dr R. Coffman, DNAX Research
Institute, Palo Alto, CA), and an erythroid marker (TER-119,I8
obtained from Dr T. Kina, Kyoto University, Kyoto, Japan). Cells
expressing any of these markers were removed by two cycles of
immunomagnetic depletion as described above. Residual positive
cells were labeled by staining with a fluorescent antirat Ig (Caltag).
After a blocking step of 10 p,g rat Ig (Pel-Freez, Rogers, AR) for 5
minutes, cells were stained with phycoerythrin-conjugated anti-Ly6A/E (E13 161-7, conjugated to phycoerythrin using reagents
obtained from Molecular Probes, Inc, Eugene, OR) and a biotinylated anti-Thy-1 reagent. These included anti-Thy-1.2 (53-2.113
and 5a8, obtained from Caltag), anti-Thy-1.1 (19XE5I9), and
anti-Thy-1 framework (31-1lZ0).The cell suspension was subsequently reacted with streptavidin-Red613 (Immunoselect, Life
Technologies, Inc, Grand Island, NY), washed, and filtered through
nylon mesh (pore size 88 pm, Small Parts, Inc, Miami Lakes, FL)
before separation by fluorescence-activated cell sorting (FACS). A
FACStar I, modified for 5-parameter analysis and sorting (Becton
Dickinson Immunocytometry Systems, San Jose, CA), was used for
cell separations that were based on expression of Ly-6A/E, failure
to express high levels of any of the lineage antigens (Ly-6A/
E+Linneg),and either the presence or absence of Thy-1 expression.
Background levels of expression were determined by staining
lymph node or BM cells with MoAbs specific for B220 (RA3-6B2)
or CD3 (KT3-1.1) and were defined by a gate that included greater
than 95% of the peak of negative cells. Thy-l"'g cells were defined
SPANGRUDE AND BROOKS
by this gating, whereas Thy-liowcells included cells above the
background up to but not including the high level of Thy-1
expression on T lymphocytes. If reanalysis of sorted populations
indicated a level of purity less than 90% based on the original
sorting windows, the cells were sorted a second time to achieve a
high level of purity. All sorting manipulations were performed at
4°C. The number of sorted cells was confirmed by hemocytometer
counting.
Long-term repopulation assay. A competitive repopulation assay was used to assess the long-term repopulating potential of
various BM preparations. Donor and recipient animals were
congenic strains differing at the Ly-5 (CD45) gene locus. BM
recipients were exposed to 11 Gy of radiation from a I3'Cs source
(Mark I gamma irradiator; J.L. Sheperd and Associates, Glendale,
CA) at a dose rate of 1.4 Gyimin, with the total dose being
delivered in two equal fractions separated by a 3-hour rest.
Animals were maintained on aqueous antibiotics (Neomycin Sulfate, 2 mg/mL; AgriLabs, Omaha Vaccine Co, Omaha, NE) for 2
weeks after irradiation. BM cells were transferred by retroorbital
injection under methoxyflurane anesthesia, with each animal
receiving 1 x lo5 syngeneic BM cells plus 1 x lo6 immunomagnetically depleted BM cells of congenic origin. For experiments
assessing the long-term repopulating potential of populations of
hematopoietic stem cells enriched by FACS, 1 x lo2 sorted
congenic cells were transferred along with 1 x lo5 normal
syngeneic BM cells. At various times after the reconstitution,
peripheral blood was collected by retroorbital puncture and
processed for flow cytometric analysis as previously described.21
Congenic donor-derived peripheral blood cells were detected by
fluorescein-conjugated MoAbs specific for the relevant allele of the
Ly-5 molecule (clone A20.1 for Ly-5.1, clone 104.2 for Ly-5.2, both
MoAbs kindly provided by Dr Shoji Kimura, Memorial Sloan
Kettering Cancer Center, New York, NY) and were phenotyped as
T lymphoid, B lymphoid, or myeloid by staining with biotinylated
reagents specific for either CD4 (GK 1.522)plus CD8, B220, or
Mac-1 plus Gr-1, respectively. The biotinylated reagents were
detected by staining with streptavidin-phycoerythrin (Biomeda,
Foster City, CA), and dead cells were excluded from analysis by
propidium iodide staining. Two-color flow cytometric analysis of
10,000 cells from each stain allowed direct evaluation of the
relative contributions of the competing BM populations to the
circulating pool of mature cells. At the termination of competitive
repopulation experiments, the animals were killed and a similar
analysis was performed on BM suspensions.
Pre-CFU-S assay. The pre-CFU-S assay detects very early
stage cells in hematopoiesis which, unlike most CFU-S, have some
ability to ~ e l f - r e n e w .It~ ~is useful for evaluating repopulating
potential in a short-term assay, and in cases where strains of mice
are to be tested for which no Ly-5 congenic strain exists. For these
assays, 1 x 106 to 1 x 107 normal or immunomagnetic particledepleted BM cells or 2 to 3 x lo3 FACS-selected cells were
transferred into lethally irradiated recipients. Thirteen days later,
BM suspensions were prepared from these animals and transferred
into secondary groups of irradiated animals. Usually each BM
suspension (pooled from two primary recipients) was transferred
into two or three groups of four mice, with each group receiving a
fivefold dilution of the previous group's marrow dose. Thirteen
days later, the secondary groups of animals were killed and their
spleens were removed and k e d in Telleyesniczky's solution (70%
ethanol-acetic acid-formalin, 20:l:l by volume) before macroscopic surface colony count. The resulting colony counts were
averaged and the data were calculated to reflect the number of
CFU-S per femur of the primary recipient animal per lo3 FACSseparated cells or lo6 BM cells injected.
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PHENOTYPE OF MOUSE STEM CELLS
1959
RESULTS
The majoriy of long-term repopulating cells express Ly6AIE. While it is clear that one can enrich hematopoietic
stem cell activity among Ly-6A/E+ cells? the possibility of
Ly-6A/Encghematopoictic stem cells cannot be excluded.
To determine whether the Ly-6A/E molecule is expressed
by most long-term repopulating cells or by only a subset of
them, experiments were performed in which immunomagnetic particles specifically removed BM cells bearing the
Ly-6A/E surface marker. As a control, BM cells expressing
CD5 were depleted in parallel samples. These dcpleted
populations of cells were then tested in two hematopoietic
assay systems, competitive repop~lation2~-*~
and pre-CFUS.3.23.27
As shown in Fig 1, depletion of Ly-6A/E-bearing
cells markedly depressed the ability of the resulting BM
population to competitively repopulate lethally irradiated
recipients, even when the competing population consistcd
of 10-fold fewer normal cells. In contrast, CDS-depleted
marrow under the same competitive conditions provided
the majority of the long-term repopulating activity over the
entire 16-week time span of the experiment. This result was
observed whether total peripheral blood leukocytes or only
circulating myeloid cells were assayed for donor or host
origin. While the Ly-6A/E-depleted population produced
a significant number of myeloid and nonmyeloid progeny
detectable 4 weeks after the transplant, the contribution of
these cells to the peripheral blood steadily decreased ovcr
the subsequent 12 weeks. The increase in donor-derived
myeloid cells seen at the 16-week time point in Fig 1, when
BM cells were analyzed, is most likely caused by the higher
frequency of myeloid cells in BM compared with peripheral
blood rather than a trend of increasing donor contribution
to myeloid differentiation at this late time point. These
results indicatc that the majority of long-term repopulating
activity present in the bone marrow of the tested mouse
strains resides among Ly-6A/E+ cells, in agreement with
the data of Uchida and Weissman.ZH
The CD5- and Ly-6A/E-depleted populations were also
tested using the pre-CFU-S assay. As shown in Table 1, this
relatively short-term assay generally reflected the results
obtained with the same two depleted populations in the
long-term competitive repopulation assay. The number of
pre-CFU-S was dramatically reduced in BM preparations
depleted of Ly-6A/E+ cells, whereas CD5-depleted marrow contained fourfold to fivefold more pre-CFU-S activity
relative to normal marrow. The enrichment of activity after
CD5 depletion may have been caused by depletion of
surface Ig+ B lymphocytes and CDS+ T cells by the anti-Ig
immunomagnetic particles, or may indicate a negative
regulatory influence that is removed by depletion of the
lymphoid cells. In contrast to the pre-CFU-S data, Ly-6A/
E-dcpleted BM contained the same number of primary
CFU-S as did normal marrow (Table 1). However, the
primary colonies produced by Ly-6A/E-depleted marrow
were consistently smaller than those generated by the other
two populations of cells (data not shown). These observations arc consistent with the notion that the CFU-S assay
measures late-stage progenitors in addition to more primitive cells, and that these late-stage cells do not necessarily
express the Ly-6A/E antigen. The smaller size of spleen
colonies generated by LydA/E-depleted BM is consistent
with in vitro observations of high proliferative potential
colony-forming ~ells.2~
CD5 depletion resulted in an increase in CFU-S frequency, although not to the same
COS
Depleted
4 weeks
Sca-1
Depleted
8w-b
I
'
r
0
12 weeks
0 16weeks
1
I
r
I
20
40
60
80
Percent Donor Cells
Total PBL
100
0
.
,
.
20
.
40
0
0
4 weeks
8 weeks
12weeks
16weeks
.
#
60
.
,
80
.
100
Percent Donor Cells
Myeloid Only
Fig 1. Competitive repopulation by Ly-6A/E-depleted or control-depleted BM cells. After immunomagneticdepletion of cells expressing
either Ly-GA/E or C D 5 . W depleted cells were injected along with 105 normal EM cells into lethally irradiated recipient animals. The normal cells
and the recipient animals were genotypicallyLy-5.2, while the depleted populations were derived from Ly-5.1 donor animals. At the indicated
times after BMT, peripheral blood was analyzedfor the presence of donor-derived cells by flow cytometric analysis, using MoAb specific for the
two alleles of Ly-5 to discriminate donor from host cells. Donor-derivedmyeloid cells were identified in two-color analyses using the Ly-5 allelic
MoAb in combinationwith MoAbs specific for myeloid cells. The data represent the results of one of four experiments, with four animals being
representedin each group. Error bars represent standard deviationsfrom the mean. The final analysis at 16 weeks is a staining of BM cells rather
than peripheralblood.
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1960
SPANGRUDE ANDBROOKS
Table 1. Pre-CFU-S and CFU-S Content of BM Cells After Depletion
With Anti-Ly-GA/E or Anti-CD5 MoAb
Group
Normal BM
CD5 depleted
Ly-GA/E depleted
CFU-S/Femur/lOE Cells*
(pre-CFU-S)
50,63, NDS
190,350,180
3.1, <0.3, < 1.0
CFU-S/S x lo4 Cells1
(day 13 CFU-S)
6 2 3
12 2 5
724
'Primary animals for pre-CFU-S determinations received 106 cells
per mouse (normal BM and CD5 depleted) or lo7 cells per mouse
(Ly-GA/E depleted). Thirteen days later, BM cells were harvested and
transferred into secondary groups of animals for CFU-S determinations.
tBM cells treated as indicated were injected into lethally irradiated
mice at a dose of 2.5 to 5 x 1O4 cells per mouse for CFU-S determination
at day 13. Results are shown as the mean t SD combined from three
separate experiments.
*Each number represents the average value obtained from three to
six secondary recipients in a single experiment. ND, not determined.
magnitude as was seen in the pre-CFU-S assay of the same
population of cells.
Thy-I antigen expression by long-tetm repopulating cells.
To test whether long-term repopulating cells are exclusively
included among those BM cells that express the Thy-1
antigen, Ly-6A/E+LinnegBM cells were separated into two
populations based on negative or low expression of the
Thy-1 antigen (Fig 2). Isolated populations of cells expressing negative or low levels of the Thy-1 antigen were tested
by both long-term competitive repopulation and short-term
pre-CFU-S assays. By both assays, a striking difference was
noted when comparing mouse strains expressing the Thy1.1 allele with those expressing the Thy-1.2 allele. As shown
in Fig 3A for the four animals from each group that had the
greatest levels of donor-derived cells, competitive repopulating activity was present among Thy-l.lIow but not Thyl . l n e g cells. While six of six animals repopulated with 100
Thy-l.llowcells plus los normal BM cells contained greater
than 5% congenic-derived myeloid cells in peripheral blood
lymphocyte preparations at some point during the time
course of the study, only 1 of 10 animals reconstituted in a
similar competitive situation with Thy-l.lnegcells contained
greater than 5% congenic-derived myeloid cells. No animals in the group of 10 mice administered Thy-l.lnegcells
contained congenic-derived myeloid cells 20 weeks after
the reconstitution. In contrast, two of six mice administered
Thy-l.llowcells contained a large (> 60%) congenic contribution to the circulating myeloid compartment 20 weeks
later.
Very different results were observed in recipients of lo5
normal syngeneic BM plus 100 congenic cells of either the
Th~-1.2"~g
or Thy-1.2IoWphenotype. In each experimental
group, 5 of 10 animals contained congenic-derived myeloid
cells greater than 5% at some point during the time course,
and in each group several animals continued to score
positive 20 weeks after reconstitution. The majority ( > 80%)
of the circulating myeloid cells in two of these animals were
descendants of the 100 sorted congenic cells (Fig 3B).
Similar results were obtained when total nucleated peripheral blood cells were analyzed (data not shown); however, it
is likely that long-term myeloid repopulation better reflects
continuous input from a primitive progenitor because of the
relatively rapid turnover of myeloid cells compared with
lymphoid ce11s.28,30.31
The results of pre-CFU-S assays performed on the
Thy-lnegand Thy-lloWpopulations of Ly-6A/E+Linnegcells
from various strains of mice are shown in Table 2. In
agreement with the long-term competitive reconstitution
assay, very little activity was observed among Thy-l.lneg
cells derived from two different strains of mice, while
Thy-l.2""g populations from four mouse strains expressed
as much or more activity than Thy-1.2Iow populations.
Furthermore, the recovery of Thy-lIowcells from Thy-l.2-
1
I
Fig 2. Separation of Ly-6A/E+LinnegBM cells into Thy-1.ln.g and Thy-l.llow populations. (A) BM cells derived from C57BL/Ka-Thy-l.l mice
were isolated on the basis of Ly-GA/E expression in the absence of high levels of lineage markers and were subsequently analyzed for expression
of Thy-1.1. The broken line indicates background staining determined with an irrelevant antibody. The vertical lines indicate gates that defined
Thy-1.1"- and Thy-1.11oW. (B) Reanalysis of the isolated Thy-l.l"eg and Thy-1.1'OW populations after FACS separation. The two populations
generally overlapped by less than 5%.
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PHENOTYPE OF MOUSE STEM CELLS
1961
A. Thy-1.1
B. Thy-1.2
1
t
n
Fig 3. Competitive repopulating activity of various FACS-sorted populations. Lethally irradiated
C57EL/&Ly-5.1-Pepa mice received 105 normal syngeneic EM cells plus 100 sorted LydA/E+Lin%y1- or Thy-l& cells derived from Ly-5.2 donors that
expressed either the Thy-1.1 allele (C57BL/Ka-Thy1.1) or the Thy-1.2 allele (C57BL/Ka). The graph
shows t h e four animals from each group that had the
greatest levels of donor-derivedcells. Each individual
animal is shown with peripheral blood analysis for
donor-derived myeloid cells at three time points after
BMT. In addition, the number of animals in each
group that had a donor-derived population exceeding 5% of the total peripheral blood mononuclear
cells at any time point is indicated.
5
z
1
5weeks
4
8
UJ
I
I
2
UJ
8
s1
z
2
LOW
Low
5110 > 5%
3
3
4
4
0
20
40
60
80
100
Percent Donor Myeloid Cells
expressing mouse strains was significantly lower than that
from Thy-1.l-expressing strains; 59.4% 2 8.7% of Ly-6A/
E+LinnCg
cells derived from Thy-1.l-expressing strains were
among the Thy-1’’””subset, compared with 23.5% 2 6.9%
derived from Thy-1.2-expressing strains. When the percentage of total pre-CFU-S activity recovered in the Ly-6A/
E+Linnegfraction was broken down into the absolute
percentage of activity included in the Thy-1Iw population,
98.7% 2 2.4% of the activity was recovered in Thy-1.1 mice
while 19.2% -c 11.7% was recovered in Thy-1.2 mice. Thus,
whereas the data shown in Table 2 indicate that the two
subsets of cells isolated from Thy-1.2-expressing strains
contain similar amounts of activity, consideration of the
recovered number of each subset argues that the majority of
pre-CFU-S activity within the Ly-6A/E+Linnegpopulation
of Thy-1.2 mice is contributed by cells that lack detectable
expression of Thy-1.
Repopulating potentid of Thy-1.2”q cells is not caused by
antibody staining differences. Because two different MoTable 2. Hematopoietic ReconstitutingActivity of Thy-l Subsets
From Various Mouse Strains
CFU-SIFemurllB Cells
Mouse Strain
Thy-1 Allele
Negative
LOW
C57BL/Ka-Thy-l.l
AKRlJ
C57BLIKa
C57BLIJ-Ly-5.1-PepJb
SJL/J
DBA12
Thy-1.1
Thy-1.1
Thy-1.2
Thy-1.2
Thy-1.2
Thy-1.2
0.5 2 0.4
2.1 f 1.6
75 f 21
17 f 5
132 2 73
189%13
70 5 36
41 f 19
92 f 22
28 2 8
44 f 24
150550
Ly-GA/E+Lin- BM cells separated from the indicated mouse strains
into Thy-1- and Thy-1” subsets as shown in Fig 2 were injected
intravenously at a dose of 2 x 1oJ cells into lethally irradiated mice.
Thirteen days later, BM was harvestedfrom those mice and transferred
into secondary recipients for CFU-S determinations. Values represent
mean f SD from four to seven secondary recipients in representative
individual experiments.
0
20
40
60
80
100
Percent Donor Myeloid Cells
Abs were used in these experiments to separate Thy-l.1and Thy-1.2-expressing BM populations, it is possible that
differences in species, subclass, or staining specificity between the two MoAbs accounted for the observed difference in biologic activity inherent in the Thy-lnCgpopulations. A direct comparison of the Thy-lnegand Thy-1’’””
populations isolated with each of the MoAbs showed that
the final populations were very similar in terms of the level
of Thy-1 expression (data not shown). In addition, staining
of lymph node T lymphocytes by the two MoAbs showed
that the anti-Thy-1.2 MoAb stained these cells about
twofold brighter than did the anti-Thy-1.1 MoAb, arguing
that the failure to recover long-term repopulating activity
exclusively in the Thy-1.2”””population was not because of
staining problems intrinsic to the MoAb.
The variability of Thy-1 staining of BM populations with
various MoAbs is shown strated in Fig 4. Using several
MoAbs specific for the Thy-1.2 allele, Ly-6A/E+LinnCg
BM
cells were stained and the patterns of staining were compared. One MoAb (5a8) failed to detect Thy-1.2” expression among Ly-6A/E+LinnegBM cells. This MoAb also
stained T h ~ - l . 2 ~T
@cells in lymph node preparations to a
lesser extent than did the other MoAb (Fig 4). An antiframework MoAb (31-11) that can be used to stain either allele of
the Thy-1 moleculemstained Thy-1.2 lymph node T lymphocytes at roughly 70% the intensity of the allele-specific
anti-Thy-1.2 MoAb used in previous experiments (Fig 4).
The 31-11 MoAb was similar to the allele-specific 53-2.1
MoAb in staining Thy-1.2’’””cells.
To definitively address the question of whether staining
with different antibodies contributed to the observed differences in functional activity, separations of BM cells were
performed using the 31-11 antiframework MoAb to stain
Thy-1 in each of the allelic forms. The results of pre-CFU-S
assays on the separated populations (Table 3) were entirely
consistent with what had been observed in previous experi-
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1962
SPANGRUDE AND BROOKS
n
E
0.1
Control
0.1
1
10
1w
lma
1
34011
1w
0.1
53k1
5a8
Arbitrary Fluorescence Units
ments using allele-specific MoAb. Virtually all pre-CFU-S
activity detectable among Ly-6A/E+Linneg cells was included in the Thy-l.llOWpopulation, while the Th~-1.2"~g
and Thy-1.2IoWpopulations both contained significant levels
of activity.
DISCUSSION
The prospect of studying the hematopoietic process in
animal models has been greatly enhanced by the ability to
identify and enrich primitive hematopoietic stem cells by
virtue of MoAb staining of cell-surface antigen^.^,^,^^ However, to embark on any quantitative analysis of the compartment of primitive hematopoietic stem cells, eg, after treatment with myeloablative agents such as 5-fluoro~racil~~
or
exposure to various infectious agents, some certainty must
be invested in the phenotypic characteristics of the hematopoietic stem cell compartment. For this reason, we have
carefully evaluated the functional characteristics of BM
Table 3. HematopoieticReconstitutingActivity of Thy-1 Subsets
Isolated Using Several MoAbs
CFU-S/Femur/103 Cells
Mouse Strain
MoAb
Negative
Low
C57BL/Ka-Thy-l.l
19XE5
31-11
53-2.1
31-11
1.7 2 0.7
<0.6
7 5 % 21*
80 2 14
45 22
75 22
92 % 22"
138 2 52
C570LlKa
1w
*
BM subsets were isolated from the indicated mouse strains using
eitherthe allele-specific MoAb 19XE5 (anti-Thy-1.1) or 53-2.1 (anti-Thy1.2) orthe antiframeworkThy-1 MoAb 31-11. Lethally irradiated primary
recipient animals received an intravenous injection of 2 x l o 3 cells of
the indicated subset. Thirteen days later, BM was harvested for CFU-S
determination in lethally irradiated secondary recipients. Values represent the mean % SD obtained from individual experiments, with each
number representing three to eight spleens from secondary recipients.
'Values are from Table 2.
1
1MO
Fig 4. Comparison of Thy-1 detection by three
MoAbs. Ly-GA/E+LinWBM cells were enriched from
C57BL/Ka mice and evaluated for staining of Thy-1
by the antiframework MoAb 31-11 and the two
allele-specific MoAbs 5a8 and 53-2.1. The control
panel shows staining with an irrelevant MoAb directed against CD3. Broken lines indicate staining of
lymph node T cells by the indicated MoAb. Mean
fluorescence intensities of the T-cell stains were 47.5
arbitraryfluorescenceunits (AFU) for 31-11,71.3 AFU
for 53-2.1, and 24.1 AFU for 5aS. Note the failure of
5a8 to detect Thy-1 staining of BM above the background level, which is indicated by the vertical lines
in each panel.
cells after separation into subpopulations based on MoAb
staining, using two assay systems (competitive repopulation
and pre-CFU-S) to detect primitive hematopoietic repopulating cells. The pre-CFU-S assay has previously been
shown to be an excellent short-term in vivo assay in terms of
correlation with long-term repopulating potential,11,23 a
result substantiated by the experiments shown here. Therefore, the pre-CFU-S assay has great potential as a quantitative approximation of long-term repopulating potential in a
short-term assay and offers the ability to screen putative
hematopoietic stem cell populations before assay in the
long-term competitive repopulating assay.
Both the competitive repopulation assay and the preCFU-S assay provided compelling evidence to suggest that,
while a low frequency of Ly-6A/Eneghematopoietic stem
cells may exist, the majority of stem cells in the analyzed
mouse strains express the Ly-6A/E antigen (Fig 1 and
Table 1). This observation is only valid for mouse strains of
the Ly-sb haplotype (eg, A m , C57BL, DBA, SJL) because
Ly-6A/E is not expressed to any great extent by cells from
mice of the Ly-ba haplotype without prior stimulation of the
~ e l l s .Therefore,
~ ~ , ~ ~ this cell surface marker has a limited
application for mouse hematopoietic stem cell enrichments, because in many strains of mice it is not expressed
on the population of interest (Spangrude GJ, Brooks DM:
in preparation).
The ideal MoAb for enrichment applications such as
those described here would have wide reactivity to hematopoietic stem cells of all mouse strains; several MoAbs have
been described as being useful for stem cell enrichments3638; however, no broad screen of many mouse strains has
been reported to date for these MoAbs. Lectins such as
wheat germ agglutinin have been useful for stem cell
enrichment^^.^^ and would be less likely to display species
variability; however, populations enriched by this method
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1963
PHENOTYPE OF MOUSE STEM CELLS
include relatively mature day 8 CFU-S unless additional
selective criteria, such as rhodamine-123 staining, are also
applied.32 The need for multiple selective criteria is a
common feature of all stem cell enrichment protocols
because no MoAb has been described that reacts exclusively with hematopoietic stem cells.
In the stem cell enrichment protocol used in these
studies, the multiple selective criteria included Ly-6A/E
and Thy-1 antigen expression as previously described.2J1
However, evaluation of Thy-1 expression by BM cells active
in the competitive repopulation and pre-CFU-S assays
produced a surprising result. By both assay systems, BM
cells derived from Thy-1.2 genotype mice included significant activity among Th~-1.2"~g
as well as Thy-1.2Iow cells
(Table 2, Fig 3). In contrast, Thy-1.1 genotype mice
included virtually all activity in the Thy-l.llo" population, in
agreement with a previous study.28One possible explanation for this observation is that the MoAb used for
separating the populations based on Thy-1.2 expression
does not stain the T h ~ - 1 . 2 ~population
~"
brightly enough to
distinguish low-level staining from negative staining. However, the experiment that uses the antiframework MoAb to
separate Thy-lneg and Thy-llO" cells from mouse strains
expressing either allelic variant of Thy-1 (Table 3) eliminates the possibility that differences in MoAb staining
account for the inability to isolate all hematopoietic repopulating activity in the Thy-1.2IoWpopulation, and argues for
a difference in the expression or configuration of the Thy-1
molecule in the mouse strains expressing the Thy-1.2 allele.
Variations in tissue-specific expression of Thy-1 determinants with dependence on the allelic variant of the molecule have been reported previously.3941
Our results are consistent with those recently reported by
Uchida and Weissman,**who used competitive repopulation experiments to show that the majority of hematopoietic
stem cell activity in one particular strain of mice (C57BL/
Ka-Thy-1.1) was included among Thy-1IoWLinnegLy-6A/E+
BM cells. We have extended those studies to show that the
concept of a universal phenotypic description of mouse
stem cells by these criteria is incorrect. From our results,
less than half of the long-term repopulating activity found
in normal BM (as estimated by the pre-CFU-S assay) is
recovered among Thy-lkoW
cells of mouse strains expressing
the Thy-1.2 allele, which includes the majority of strains. It
is likely that depletion of cells expressing lineage antigens
remains a viable approach for enriching mouse hematopoietic stem cells, because most of these antigens are not
known to exist in allelic forms and because the approach of
lineage depletion has been adopted by many different
laboratories using a wide variety of mouse strains. In
addition to the study of Uchida and Weissman,28Jordan et
aP6 have reported that virtually all hematopoietic stem cells
present in mouse fetal liver tissue fail to express high levels
of lineage markers.
The present study shows that Thy-1 expression is not a
universal characteristic of hematopoietic stem cells in the
mouse. In this regard, it is interesting to note the recent
demonstration of Thy-1 expression on cells derived from
human fetal liver that have many characteristics of hematopoietic stem cells.42It is unclear how much variation will be
observed between individual humans with regard to Thy-1
expression on these cells, and how the transition from fetal
to adult tissue will affect the phenotype of the long-term
repopulating cells. The fetal liver-derived hematopoietic
stem cell population in the mouse shares the MoAb-defined
phenotype of adult BM-derived cellsls; however, these
experiments were performed using Thy-1.1 genotype animals. Furthermore, experiments designed to trace Thy-1
and Ly-6A/E expression by all assayable long-term repopulating cells in mouse fetal tissues derived from a variety of
strains have not been reported. It is possible that the cell
surface phenotype of these cells may change during ontogeny, as during recovery from chemically induced myeloablat i ~ nTherefore,
.~~
it is difficult to predict, based on the data
currently at hand, whether hematopoietic stem cells derived from all individual human fetal or adult sources will
invariably express the Thy-1 antigen.
The demonstration here that many hematopoietic stem
cells do not express the Thy-1 antigen argues against an
obligatory role for this molecule in hematopoietic stem cell
biology, at least when the cells are in a resting state.
However, there may be redundancy in the molecules able to
fill any putative role of the Thy-1 molecule. It is interesting
to note that Muller-Sieburg et a1 found Thy-1 to invariably
be expressed on cells capable of initiating Whitlock-Witte
long-term BM cultures.43 These experiments were performed primarily in BALB/c mice, a strain that does not
express Ly-6A/E on resting hematopoietic stem cells.
Perhaps one of these two molecules is sufficient for any
necessary functional role, and in the absence of Ly-6A/E
the expression of Thy-1 may become critical. Experiments
to test whether Thy-1 is invariably expressed on hematopoietic stem cells of L Y - mouse
~~
strains, which do not
constitutively express Ly-6A/E, are currently under way.
ACKNOWLEDGMENT
We thank Drs Seth Pincus, John Portis, Donald Lodmell, and
Kim Hasenkrug for critical review of this manuscript.
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