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Characterization of Mouse Lymphohematopoietic Stem Cells Lacking Spleen
Colony-Forming Activity
By Richard J. Jones, Michael I. Collector, James P. Barber, Milada S. Vala, Mary J. Fackler, W. Stratford May,
Constance A. Griffin, Anita L. Hawkins, Barbara A. Zehnbauer, John Hilton, 0. Michael Colvin, and Saul J. Sharkis
The classical definition of lymphohematopoietic stem
cells
(LHSC), the most primitive progenitors of all bloodcells,
requires that they have the capacity for self-renewal and
for the long-term production of all blood cell
lineages.
However, other characteristics of LHSC havebeen debated. Our previous datasuggested that mouse LHSC are
very slowly proliferating
cells that generate delayedmultilineage engraftment, while ”radioprotection” (rapid engraftment that will prevent early death from radiationinduced marrow aplasia) results from more committed
progenitors. Alternatively,some
groups have reported
that mouseLHSC are responsiblefor both radioprotection
and long-term repopulation of all blood cell lineages. A
possible explanation for this difference is that cells with
the capacity for long-term production of all blood cell
lineages are biologically heterogeneous. We now show that
10 LHSC can generate all blood cell lineages for the lifetime of theanimal. However, these cells lacked radioprotection and spleen colony-forming activity. LHSC were
identified and isolated by their small
size, their lack of
expression of antigens characteristic of mature blood cell
lineages, and their high expression of aldehyde dehydrogenase. In addition, these cells were foundt o express undetectable or l o w levels of many antigens presumed t o
mark LHSC, including Thy-l, Ly-GA/E (Sca-l), c-kit, and
CD34. There appears t o be at least two classes of LHSC
with the capacity for long-term production of all blood
cell lineages: one thatgenerates both radioprotection and
long-term engraftment and
one that produces delayedbut
durable engraftment. Our data suggest that this latter
class may represent a very primitive class of LHSC.
0 7996 by The American Society of Hematology.
T
cago, IL) equipped with a potentiometer provided precisely metered
flow of medium. Male BM cells (=5 X 10’) were loaded into the
chamber at a flow rate of 15 mL/min and a rotor speed of 1,260g.
The cells were eluted by changing flow rates and keeping the rotor
speed constant, collecting about 300 mL at each flow rate! BM cells
that lack expression of antigens characteristic of mature blood cell
lineages ( h - ) were initially isolated by direct immune adherence
( ~ a n n i n g using
)~
60-mm polystyrene Petri dishes adsorbed with a
cocktail of 60 pg each of rat-antimouse AA4.1, B220 (B-lineage):
Lyt-l (CD5, T-lineage), GR-I (granulocytes), Mac-l (monocytes/
macrophages),* and TERll9 (erythr~id).~Monoclonal antibodies
(MoAbs) were provided by J.McKearn (AA4.1; Monsanto, St Louis,
MO) or I. Weissman (anti-GR-l; Stanford University, Palo Alto,
CA), or purchased from PharMingen (San Diego, CA) (anti-TER
119) or American Tissue Type Culture (Rockville, MD). The nonadherent ( h - ) cells were removed by rocking and gentle aspiration
after incubating lo7 cells/mL on the panning plates at 4°C for 90
minutes. Aldehyde dehydrogenase (ALDH) activity was assessed by
incubating lo6cells/mL at 37°C for 30 minutes in an activated dansyl
aminoacetaldehyde (DAAA) solution as previously described.’ Subsequently, the cells were incubated at 4°C for 30 minutes with the
above antibody cocktail and then incubated with bothphycoerythrinconjugated goat-antirat IgM (1:400) and IgG (1:2,000) (Southern
Biotech, Birmingham AL). Lin- (<99% of control fluorescence)
anddansyl’ (>99% of unstained cells’ fluorescence) cells were
sorted and collected.
In vivo and in vitro assays. To determine their engraftment potential, putative male LHSC were combined with 2 X lo4 female
HE PHENOTYPE AND function of lymphohematopoietic stem cells (LHSC), the most primitive progenitors
of allblood cells with the capacity for self-renewal and
the long-term production of all blood cell lineages, remain
controversial.’ Some groups have reported that mouse LHSC
are responsible for both “radioprotection” (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) and long-term repopulation of all
blood cell
These LHSC are also enriched for day
12 spleen colony-forming units (CFU-S).2,3Alternatively,
we and others have suggested that LHSC are slowly proliferating cells that generate delayed multilineage engraftment,
whereas radioprotection results from more committed prog e n i t o r ~ . Moreover,
~.~
we found that LHSC could be separated from committed progenitors, including day 12 CFUS, on the basis of their small size by counterflow centrifugal
elutriation (CCE).4 We believe that the most likely reason
for this difference is that cells with the capacity for the longterm production of all blood cell lineages are biologically
heterogeneous. We further identified and purified LHSC by
their lack of expression of antigens characteristic of mature
blood cell lineages and their high expression of aldehyde
dehydrogenase, in addition to their small size. LHSC were
then characterized functionally and by their expression of
antigens that have been reported to mark LHSC.
MATERIALS AND METHODS
Animals. B6D2Fl mice (National Cancer Institute, Frederick,
MD), 6 to 12 weeks of age, were used for all the studies. Female
B6D2Fl mice were used as transplant recipients and were housed
in sterile microisolator cages. They were fed acidified water and
sterilized lab chow ad libitum. Bone marrow (BM) from male
B6D2F, mice was used to isolate putative LHSC.
Isolation ofmouse LHSC. For each isolation experiment, 8 to
10 male mice were killed by cervical dislocation, and the hind legs
were removed. BM was flushed from the medullary cavities of the
femurs and tibias using medium and a 25-gauge needle. Single-cell
suspensions were produced by repeated passage through the needle.
CCE was performed using a Beckman J-6M centrifuge with a E6B elutriator rotor and a standard chamber (Beckman Instruments,
Palo Alto, CA). A master-flex peristaltic pump (Cole Palmer, ChiBlood, Vol 88, No 2 (July 15). 1996: pp 487-491
FromtheJohns Hopkins Oncology Center, TheJohnsHopkins
Medical Institutions, Baltimore, MD.
Submitted December 18, 1995; accepted March 20, 1996.
Supported in part by National Institutes of Health Grant No. CA
15396. R.J.J. is a Leukemia Society of America Scholar.
Address reprint requests to Richard J. Jones, MD, Room 2-127,
JohnsHopkins Oncology Center, 600 N Wove St, Baltimore, MD
212874967,
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.
0 1996 by The American Society of Hematology.
0006-497l/96/8802-0158$3.00/0
487
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JONES ET AL
400
cells from the CCE large cell or "Rotor Off" (RO) fraction, those
remaining in the chamber after eluting the smaller cells at 33 mL/
min; and transplanted via the tail vein into marrow-ablated female
B6D2Fl mice. Marrow ablation was achieved with 1,150 cGy of
total body irradiation (TBI) and 0.2 mg of dimethyl myleran (DMM)
injected intraperitoneally 24 hours before the TBI. Residual host
LHSC that survive marrow ablation compete in long-term engraftment with donor stem cells, especially when small numbers are
transplanted; the addition of DMM to TBI enhances suppression of
host LHSC.' Granulocyte-macrophage colony-forming units (CFUGM) and CFU-S were assayed as previously described?." Briefly,
the CFU-GM cultures contained 30% fetal calf serum, 1% bovine
serum albumin,
molL 2-mercaptoethanol, and 10% Wehi-conditioned medium as a source of colony-stimulating factor.
Assessment of engraftment by fluorescence in situ hybridization
(FISH)for the Y chromosome. Peripheral blood (PB) was obtained
by retro-orbital sinus puncture with a heparinized microhematocrit
tube to assess engraftment. For determination of lineage engraftment,
mice were killed by cervical dislocation. CFU-GM colonies were
cultured from the BM, plucked, and pooled (about 500 to 2,000
colonies per mouse) to quantitate myeloid engraftment. B cells were
recovered by panning plastic-nonadherent spleen cells (IO' cells/
mL) treated with anti-B220 MoAb and placed in anti-rat IgM-coated
Petri dishes for 45 minutes at 4°C. Unbound cells were removed by
gentle aspiration; the adherent B220' B cells were obtained by vigorous pipetting. T cells (thymocytes) were obtained by gently dissociating the thymus through a 60-pm mesh screen.
FISH for Y chromosome was performed using modifications of
our previously described technique.'' A mouse Y-specific probe for
interphase FISH analysis was constructed by microdissection and
subsequent polymerase chain reaction (PCR) amplification and biotinylation. Five copies of the Y chromosome q-arm were scraped
from a G-banded standard chromosome preparation of male mouse
splenocytes and amplified by PCR" using a degenerate primer.' '
The probe was biotinylated by including biotin-dUTP at 20 pmol/
L in a secondary PCR reaction, precipitated with mouse Cot-1 DNA
(Life Technologies, Gaithersburg, MD), and used at approximately
200 ng/30 pL hybridization mix (50% formamide, 10% dextran
sulfate, 2X standard saline citrate phosphate [SSCP; 0.15 mol/L
NaCI, 0.015 mol/L Na citrate, 0.02 mol/L Na,H,POJ) for FISH.
Every experiment included a standard curve of known mixtures of
male and female cells; the percent male varied by only t 1 % on
samples of 100% male cells, and false positives (signal on 100%
female cells) were never seen. This probe is a significant improvement over our previous method" because the signal is larger and
more distinct, the sensitivity and specificity are improved, and there
is no requirement for proteinase K treatment of the slides before
FISH. The percent male cells (mean t SEM) results include all the
assayed animals. Comparison of long-term engraftment at different
times after transplant was performed by analysis of variance (ANOVA).
Flow cytometry for expression of T h y 1 and Ly-6A/E (Sca-1).
After isolation, cells were allowed to rest at room temperature for
I hour. A single-cell suspension of thymocytes, as a positive control
for Thy-I expression, was obtained as described above. The CCE
large cell (RO) fraction was the positive control for Ly-6NE expression. The cells were incubated with gentle agitation for 30 minutes
at 4°C with either 1 pg of fluorescein (FITC)-conjugated anti-CD90
(Thy- 1.2) or 2 pg FITC-conjugated a n t i - L y 6 - a (PharMingen).
FITC-conjugated rat IgG2. K control antibody (PharMingen) served
as a negative control. After the incubations with the antibodies, the
cells were washed and resuspended. Antibody fluorescence intensity
was measured on a FACScan flow cytometer (Becton Dickinson,
Palo Alto, CA). The results reported in the text are the mean t
SEM of at least five experiments.
Reverse transcriptase (RT)-PCRfor expression of c-kit or CD34.
We used our previously described pr~cedure'~.''
with minor moditications. Total RNA was prepared using the REX total RNA Extraction Kit (USB, Cleveland, OH). To eliminate DNA contamination,
the RNA was treated with DNAase I (FPLC pure; Pharmacia, Alameda, CA). First-strand DNA was synthesized with 300 U Moloney
Murine Leukemia Virus RT (Gibco BRL, Gaithersburg, MD) using
2-ng random hexanucleotides (Pharmacia) as primers" in the presence of 4 U RNasin (Promega, Madison, WI). The cDNA product
was divided for experimental (c-kit or CD34) or control (mouse pactin) PCR reactions. The sequences of the CD34 primers'" and
the mouse @-actinprimers'"'' have been previously published. The
sequences of the mouse c-kit primers arela: 5' sense (1-24) (S'GAGCTCAGAGTCTAGCGCAGCAC-3') and 3' antisense (739A Coy temp7 16) (5'-TTTTATGGTGCACACCACCGTAAA-3').
cycler (Model 50; Coy Laboratory Products Inc, Grass Lake, MI)
was used with the following program: 5 minutes at 95°C; 35 cycles
at 55°C for I minute, 72°C for I minute, 95°C for 1 minute, and
one final 72°C extension for S minutes. The amplified products were
analyzed by electrophoresis and staining by ethidium bromide. To
ensure specificity of RT-PCR for c-kit and CD34 beyond mobility
of the product on the gel, the products were transferred and hybridized with either a '*P-labeled c-kit-specific probe (5'-CAATGGCCTCACGAGTTCTA-3')'8 or a CD34 exon 3-specific probe (S'ACATCACCCACCGAGCCATA-3' ) . I h
RESULTS
We previously showed that small mouse BM cells isolated
by CCE at a flow rate of 25 mL/min (FR25) are enriched
for LHSC that produce delayed multilineage engraftment
without radiopr~tection.~
The FR25 cells represented 20%
5 5% of the whole marrow. Marrow cells that lack expression of antigens present on mature blood cell lineages (lin )
are also enriched for LHSC.' AA4.1, an antigen that is expressed by most mouse hematopoietic progenitors and B
cells," also was found to be expressed by LHSC from mouse
fetal liver2"and 5-fluorouracil-treated adult BM." However,
we found that only the FR25 lin- BM cells that were AA4.1
(0.3 2 0.1% of whole marrow) produced long-term engraftment, whereas the small AA4.l+lin- cells were unable
to produce long-term engraftment (data not shown), as others
have also reported.2221
Indirect evidence suggests that LHSC express high levels
of ALDH,2425 a n enzyme responsible for the conversion of
vitamin A to its active metabolite retinoic acid2"' and for
cellular resistance to cyclophosphamide.** We further enriched for LHSC by isolating small AA4.1-lin- cells that
highly expressed ALDH, as we previously described.' The
FR25 AA4.1-lin-ALDH+ cells were 0.005% 5 0.001% of
the whole marrow, allowing 1 to 2 X lo4 of these cells to
be recovered in one experiment using 8 to 10 mice. For
transplantation studies, these cells were combined with a
distinguishable source of cells that provide radioprotection:
the CCE large cell (RO) fraction that is enriched for committed progenitors while depleted of LHSC." When transplanted
into marrow-ablated female B6D2FI mice (20 mice from
four separate experiments) with 2 x lo4 female CCE RO
BM cells: 10 male FR25 AA4.1-lin-ALDH' BM cells were
able to generate most of the PB cells present in the mice for
up to 15 months (Fig 1). Only 4 of 20 mice died; there were
2 early deaths (at 21 and 28 days after BMT) and 2 mice
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CHARACTERIZATION OF MOUSE STEM CELLS
Survival
16/20
18/20 18/20
0'
0
489
8/12
I
3
9
6
Months After
12
15
BMT
Fig 1. Engraftment of mice transplanted with 10 FR25 AA4.1-lin-ALDW BM cells. Follow-up was 15 months for two experiments
(12 mice) and 9 months for two experiments (8 mice).
died late (at 8 and 9 months) after BM transplantion (BMT)
of unknown causes. The engraftment from the 10 small
AA4.1-lin-ALDH+ BM cells was durable (Fig 1); the fraction of PB cells derived from these cells at 4, 9, and 15
months after BMT (92% 2 3%, 83% 2 5%, and 68% t
lo%, respectively) was not significantly different (P = .15,
ANOVA). Moreover, all blood cell lineages (myeloid, B
cell, T cell) were derived from the 10 small
AA4.1-lin-ALDHf cells (Table 1). All the long-term repopulating activity was in the ALDH' cells, as 14 of 16 female
mice (from three separate experiments) transplanted with
1,000 small AA4.1-lin-ALDH- male BM cells (combined
with 2 X lo4 female CCE RO cells to provide transient,
early engraftment) died 21 ? 1 days after BMT; the two
Tabla 1. In Vitro and In Vivo Analysis of Small
AA4.1-lin-ALDH' Calls
No. of Cells Assayed
NO. Of CFU-GM
NO. Of d 12 CFU-S
1,000
10
0.125 f 0.006
0.08 2 0.07
-
Survival after BMT*
0116
16/20
(9 mol
% Donor engraftment
(9-15 mo after
BMT)
CFU-GM
B cells
-
96 ? 1
92 5 2
89 ? 3
T cells
-
Four mice (2 mice 9 months and 2 15 months after BMT, from two
separate experiments) were killed to analyze the specific lineages for
origin of engraftment.
* Transplants for survival with 10 cells were combined with 2 x 10'
female CCE RO marrow cells to provide a transient, early wave of
engraftment4; 1,000 cell transplants received no RO cells. Mice receiving 1,000 cells died at a median of 15 5 0.5 days after BMT.
mice that survived were completely repopulated with female
cells at 180 days after BMT.
Despite being able to generate all blood cell lineages for
the lifetime of the animal, the small AA4.1-lin-ALDH+ BM
cells were unable to provide radioprotection. All mice (16
of 16 from three separate experiments) transplanted with
1,000 FR25 AA4.1-lin- ALDH' cells, but no CCE RO cells
to provide initial engraftment, died 15 t 0.5 days after BMT
of BM aplasia (Table 1). Moreover, mice receiving transplants of 1 X lo4 small AA4.1-lin-ALDH+ marrow cells
(and no CCE RO cells) also died of aplasia (2 experiments).
The small AA4.1-lin-ALDH' marrow cells contained almost no CFU-GM or the more primitive day 12 CFU-S
(Table l), which some groups have found to be inseparable
from LHSC.'*3 There were only a total of two CFU-GM
colonies in the 16 plates (1,000 FR25 AA4.1-lin-ALDH'
cells cultured per plate) from four separate experiments. Similarly, only one spleen colony was observed in 12 mice (from
three separate experiments) injected with 1,000 FR25
AA4.1-lin-ALDH+ cells per mouse.
The small AA4. 1-lin-ALDH' cells were also capable of
in vivo self-renewal as assayed by serial BMT.' BM was
obtained from four mice that had been transplanted with 10
FR25 AA4.1-lin-ALDH+ BM cells (2 mice were 9 months
after BMT and 2 from a separate experiment were 15 months
after BMT), and lo6 cells were transplanted into 4 marrowablated secondary female recipients for each primary recipient. Of the 16 secondary recipients, 14 survived 4 months
and 10 are alive 8 months after BMT with 31% t 8% and
22% t 5% of their PB cells at 4 months and 8 months,
respectively, derived from the primary transplant of 10 small
AA4.1-lin-ALDH+ BM cells. These data show that the 10
cells in the primary transplant underwent in vivo self-renewal because the secondary transplant of lo6 cells represented only about 0.3% of the BM cells present in the reconstituted primary recipient^.'^
Most antigens reported to mark LHSC, such as Thy-1,'
Ly-6AE (Sca- 1 p 3 kit,^,'* and CD34,30have been found
to be expressed on cells with the capacity to generate both
radioprotection and long-term engraftment. Because the
small AA4.1-lin-ALDH+ BM cells produced only delayed
engraftment, we examined the expression of these other antigens on these cells. Thy-1 could not be detected on these
cells by flow cytometry (Fig 2A). Similarly, c-kit expression
could not be detected by flow cytometry with anti-c-kit
MoAb (data not shown) or by RT-PCR for mRNA expression (Fig 3). In addition, the c-kit RT-PCR results were
confirmed with multiple other sets of primers (data not
shown). Ly-6AE was expressed on 7.2% t 2.5% of the
small AA4.1-lin-ALDH+ BM cells by flow cytometry (Fig
2B). Although CD34 expression was detectable in the small
AA4.1-lin-ALDH' BM cells by RT-PCR, the levels were
lower than those seen in the CCE large cell fraction (Fig 3)
that is enriched for less primitive hematopoietic progenitors
(ie, CFU-GM and CFU-S).4DAAA staining did not influence
antigen expression; the FR25 AA4.1-lin- cells (not sorted by
ALDH expression) displayed similar low-level expression
of Ly-6A/E, and c-kit expression was undetectable by RTPCR.3'
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JONES ET AL
490
Thv 1.2
L
- Small AA4nin- ALDH+
-
a -
Antibody Control
Fluorescence
SCA-1
- Small AA4nin- ALDH+
Antibody Control
-- CCE Large Cells
5:
n E
3
-
z
- --
s :-
---
DISCUSSION
The classical definition of LHSC requires that they have
the capacity for self-renewal and for the long-term production of all blood cell lineages.’ However, this definition may
CKlT
ACTIN
CKlT
M SCRODW SC RO SC RO DW
CD34
not be specific for the most primitive progenitor of blood
cells. These criteria for LHSC appear to be fulfilled by at
least two classes of cells: one that generates both radioprotection and long-term engraftment (and expresses most antigens
presumed to mark LHSC),’.3 and one that produces delayed
but durable engraftment (and expresses undetectable or low
levels of AA4. I , Thy- I , Ly-6A/E, c-kit, and CD34). Some
antigens (eg, Thy-I ,“ Ly-6A/E3’) have been shown to be
expressed by LHSC from some normal strains of mice but
not other strains. Although mouse strain variability may explain some of the phenotypic differences of LHSC, it probably does not explain the functional differences. Further, antigens that are not invariant characteristics of LHSC from all
normal mouse strains are unlikely to be biologically essential
for LHSC function.
Identifying the most primitive LHSC with “unlimited”
proliferative potential is essential if they are to be used in
clinical transplantation, especially as treatment of inherited
diseases such as the hemoglobinopathies. The transplantation
of LHSC with limited proliferative and self-renewal potential
may result in late donor graft failure, perhaps even years
after BMT. It is possible that late donor graft failure may
not be critical in the setting of a malignancy, where BMT
is primarily a rescue procedure for marrow-ablative doses
of cytotoxic therapy aimed at the malignancy. Rapid, early
engraftment is the primary goal of BMT for malignancy so
that the complications of aplasia can be averted. Most BMT
preparative regimens do not appear to be truly marrow-ablative, as shown by the frequent occurrence of at least partial
host hematopoiesis after T-cell-depleted allogeneic BMT.
Therefore, late donor stem cell failure after BMT for a malignancy would be of no significance if residual normal host
hematopoiesis returned. However, in the setting of an inherited disease, eventual graft failure resulting from the transplantation of LHSC with limited proliferative and self-renewal potential would lead to resurgence of an abnormal
genetic phenotype even if host hematopoiesis returned.
Engraftment of all blood cell lineages arising from limiting numbers of LHSC that also are capable of radioprotection and producing day 12 CFU-S appears to significantly
decrease with time after primary BMT, and particularly after
serial BMT.’.’4.3s Conversely, limiting numbers of small
AA4.1 -lin-ALDH’ BM cells produced sustained engraftment of all blood cell lineages for the lifetime of the
ACTIN
M SC RO DW SC
RO
CD34
SC
RO DW
-
-7
Fig 3. RT-PCR for expression
of c-kit or CD34 by 10‘ small AA4.1-tin-ALDH’ BM cells ISCI. 10‘
cells from the CCE RO fraction
served as positive controls. Although CD34 expression by
small AA4.l-Iin’ALDH‘
cells
was not detectable on ethidium
staining (dark panels], it was apparent after hybridization (light
panels). These data were confirmed in at least three separate
experiments. M, 100-bp size
markers; DW, distilled water.
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CHARACTERIZATION OF MOUSE STEM CELLS
mouse, and in secondary recipients upon serial BMT. Thus,
the small AA4.1-lin-ALDH+ BM cells, which exclusively
generate delayed engraftment and lack spleen colony-forming activity, appear to represent a very primitive class of
LHSC.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1996 88: 487-491
Characterization of mouse lymphohematopoietic stem cells lacking
spleen colony-forming activity
RJ Jones, MI Collector, JP Barber, MS Vala, MJ Fackler, WS May, CA Griffin, AL Hawkins, BA
Zehnbauer, J Hilton, OM Colvin and SJ Sharkis
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