1. Art and Diseño

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Enrichment and Functional Characterization of S c a - l + W G A + , Lin- W G A + ,
L i n - S c a - l + , and L i n - S c a - l + WGA+ Bone Marrow Cells
From Mice With an Ly-6" Haplotype
By Roland Jurecic, Nguyen T. Van, and John W. Belmont
Approximately 4%t o 5% of all bone marrow (BM) cells and
8%to 9%of low density BM cells from FVB/N and BALB/c
mice (Ly-Sahaplotype) show high t o intermediate expression of Ly-6E.1 antigen, recognized by the Sca-I antibody.
Functional properties of enriched cells expressing Ly-6E.1allelic form of Sca-I antigen were analyzed and correlated
with the properties of cells expressing the carbohydrate
binding sites for the lectin wheat-germ agglutinin (WGA).
Using equilibrium density centrifugation and fluorescenceactivated cell sorting, Sca-1 +WGA+, Lin-WGA', LinSca-1 +,and Lin-Sca-1 +WGA+ cells were isolated and
their splenic colony-forming unit (CFU-S) cell content, radioprotection ability, and long-term reconstitution capacity
determined. Enriched Sca-1 WGA+, Lin-WGA+, LinSca-I and Lin-Sca-1 +WGA+ cells gave rise to 1 CFUS12cell out of 26,20,21, and 1 5 sorted cells, respectively.
When transplanted into lethally irradiated recipients
(100 t o 500 cells/mouse) all populations rescued 70%to
100% of recipients in a 30-day radioprotection assay and
mediated survival of 40% t o 80% of recipients 6 months
after transplantation. Using transgenic mice as cell donors
w e have shown that 1 2 to 16 weeks after transplantation
of 100 Sca-1 +WGA+, Lin-WGA+, Lin-Sca-l+, and
Lin-Sca-I WGA+ cells, 40%to 80%of recipients had donor cells in BM, spleen, thymus, and lymph nodes. These
results indicate that the population of cells expressing Ly6E.1 form of Sca-1 antigen in t w o analyzed mouse strains
with Ly-6" haplotype contains CFU-S and long-term repopulating cells. Furthermore, the data suggest that, at least
in FVB/N mice, day-I2 CFU-S cells and cells with longterm repopulating capacity simultaneously express Ly6E.1 form of Sca-I antigen and WGA-binding molecules.
0 1993 by The American Society of Hematology.
H
nonstimulated but strongly expressed on stimulated T
ceIls.20,21
A population of Thy- 1'"Lin-Sca- 1' cells, isolated from
C57BL mice, was shown to be approximately 1,000 times
enriched for CFU-S and pre-T activity. Small numbers of
these cells can generate long-term repopulation of both lymphoid and myeloid lineages, can protect mice from lethal
irradiation, and can further repopulate secondary recipients, indicating self-renewal ~ a p a c i t y . ~ ~Recently,
~ ~ ' ~ * ~a~ * ~ *
simplified method for enrichment of mouse Lin-Sca- I + hematopoietic stem cells was e~tablished,'~
based on the fact
that it is not clear what functional activities, if any, are contributed by the Thy-1" fraction of Lin-Sca-l+ cells. Recent
studies have confirmed that Thy-1 expression is not a universal characteristic of mouse hematopoietic stem ~ e l l s . 2 ~
Further fractionation of Thy-1'"Lin-Sca- 1+ and Lin-Sca-l+
cells on the basis of rhodamine 123 retention and expression of c-kit proto-oncogene (receptor for stem cell factor)
has shown that long-term repopulating ability in Ly-sbmice
is essentially restricted to cells expressing the Sca- 1 antigen
+
+
EMATOPOIESIS is an ongoing developmental process by which large numbers of mature blood cells
with very specific functions and limited life spans are continually replenished. Experiments using retroviral integration
m a r k e d 5 have shown that all blood cell types originate
from a common population of pluripotent hematopoietic
stem cells (PHSC). In addition to ability to generate committed progenitors of the erythroid, megakaryocytic, myeloid, and lymphoid lineages, PHSC are characterized by
quiescence and capacity to ~elf-renew.~.~
A number of procedures based on physicochemical and
immunochemical characteristics have been developed for
the enrichment of murine PHSC,&" including density centrifugation, counterflow centrifugal elutriation, and fluorescence-activated cell sorter (FACS) separation based on cell
surface antigen expression, lectin binding, or uptake of fluorescent vital dyes."-" However, because there is no clonal
assay for PHSC, these studies have relied on measurement
of splenic (spleen colony-forming unit [CFU-S] cells) and
thymic colony formation, radioprotection, long-term survival of recipients, and long-term repopulation of all blood
lineages as assessed by enzyme variants, retroviral markers,
and allelic cell surface markers.5.9~10~13,16-'8
It was shownI3that mouse hematopoietic stem cells express low levels of Thy-1 antigen (Thy-I") and are lineagenegative (Lin-), ie, they do not express markers characteristic of B cells, T cells, granulocytes, and myelomonocytic
cells. A monoclonal antibody (MoAb) that recognizes stem
cell antigen-1 (Sca- I), was used to purify stem cells from the
Thy-l'OLin- pop~lation.'~
Sca-1 molecule is a member of
the Ly-6 antigen family, and its two allelic forms are expressed on hematopoietic and nonhematopoietic cells. The
Ly-6A.2 antigen is found in mice with a L Y - haplotype
~~
(C57BL, C57L, DBA, and AKR mice) and is expressed on
both nonstimulated and stimulated T cells. The Ly-6E.l
antigen is found in mice with a Ly-6" haplotype (BALB/c,
C3H, CBA, and FVB/N mice) and is expressed weakly on
Blood, Vol 82,No 9 (November l ) , 1993:pp 2673-2683
+
From the Institutefor Molecular Genetics, Howard Hughes Medical Institute, Baylor College of Medicine and the Department of
Hematology, M.D. Anderson Cancer Center, Houston, TX.
Submitted March 16, 1993; accepted July 12, 1993.
Supported by the Howard Hughes Medical Institute and Grants
No. U 0 1 A130243 and CA 16672 from the National Institutes of
Health, Bethesda, MD.
Address reprint requests to John W.Belmont, MD, PhD, Institute
for Molecular Genetics, Howard Hughes Medical Institute, Baylor
College of Medicine, Houston, TX 77030.
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 I734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8209-0019$3.00/0
2673
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2674
JURECIC, VAN, AND BELMONT
but has also shown functional heterogeneity of both
enriched
An analysis ofthe cell surface phenotype has shown that Lin-Sca- 1 cells express low levels of
heat-stable antigen, intermediate levels of phagocyte glycoprotein- l , and high levels of class-I major histocompatibility antigens (H2 K/D), leukocyte common antigen (Ly-5),
and carbohydrate binding sites for the lectin wheat-germ
agglutinin (WGA).23 Studies of long-term repopulation
(LTR) by Thy- 1'"Lin-Sca- 1+ cells have confirmed that this
population is heterogenous and that it contains in vitro colony-forming cells (CFC), day- 12 CFU-s, LTR cells for both
the myeloid and lymphoid lineages, and cells capable of
LTR of only the lymphoid
Because of a distinctive pattern of Ly-6A/E alloantigen
expression in thymocyte and T-lymphocyte subsets related
to the Ly-6 haplotype, it was suggested that bone marrow
(BM) cells from Ly-6" mice must be induced to express the
Ly-6E. 1 molecule and, thus, are not suitable for enrichment
of stem cells expressing Sca-1 antigen.23331-34
We have observed that about 4% to 5% of all BM cells and 8% to 9% of
low density BM cells from FVB/N and BALB/c mice show
high to intermediate levels of Sca-1 expression and, thus,
decided to analyze the functional and phenotypic characteristics of these cells. Furthermore, because Lin-Sca- 1+ cells
express high levels of WGA-binding molecules,23a feature
of BM cells used previously for the enrichment of CFU-S
cells,8.9, 1 1,12, I5 we wished to correlate the expression of Ly6E. 1 antigen and WGA-binding molecules on enriched BM
cell populations from Ly-6" mice. Using equilibrium density centrifugation and FACS we isolated Sca-l+WGA+,
Lin-WGA+, Lin-Sca- 1+, and Lin-Sca- l+WGA+ cells and
analyzed their CFU-S cell content, radioprotection ability,
and long-term reconstitution capacity.
FACS analysis has confirmed that the Sca-1 antibody recognizes Ly-6E. 1 antigen expressed on a small population of
BM cells in FVB/N and BALB/c (Ly-67 mice. The CFU-S
and LTR assays have shown that Ly-6E. 1+ cells include
CFU-S cells and cells with LTR ability. Furthermore, we
show that the hematopoietic stem cell activity in FVB/N
mice, as defined by LTR of hematopoietic tissues in irradiated animals, resides within a population of BM cells
coexpressing Ly-6E.l form of Sca-1 antigen and WGAbinding molecules.
+
MATERIALS AND METHODS
Mice. FVB/N and BALB/c mice were purchased from Taconic
(Germantown, NY). Transgenic FVB/N mice were generated by
DNA microinjection into FVB/N zygotes.35The microinjected
DNA was a 5.3-kb EcoRI-PstI fragment from genomic clone grl9."
The fragment consisted of a truncated SV40 large T antigen transcribed from an a-A-crystallin p r ~ m o t e r . ~All
~ , mice
' ~ were housed
in microisolator units placed on laminar-flow cage racks (Lab Products, Maywood, NJ) and fed sterilized rodent chow and sterile, acidified water.
Preparation ofBM cells. BM cells were obtained by flushing the
femoral and tibial shafts with cold phosphate-buffered saline (PBS;
CELLGRO, Hemdon, VA) supplemented with 5% fetal calf serum
(FCS; GIBCO, Grand Island, NY) and antibiotics (100 U/mL penicilin and 100 pg/mL streptomycin; GIBCO). Single cell suspension
was prepared by aspirating the clumps through a 2 1-gauge needle.
The cells were washed twice (800g for I O minutes) with PBS and
kept at 4°C throughout the whole procedure.
Discontinuous density Percoll gradient. Percoll solutions with
specific densities of 1.10, 1.09, 1.07, and 1.06 g/mL were prepared
according to the protocol recommended by the manufacturer
(Pharmacia Biotech Inc, Piscataway, NJ). The pH and osmolarity
of solutions were adjusted to pH 7.0 and 300 ? 5 mOsm/kg. All
solutions were kept sterile and at 4°C. The gradients were formed
by layering sequentially 2 mL of I. 10, I .09, and 1.07 g/mL solution
in round-bottom polystyrene tubes. On top of the gradient 2 mL of
1.06 g/mL solution was layered containing up to 5 X IO7 BM cells.
The gradient was centrifuged at 1,OOOgat 4°C for 25 minutes. Lowdensity cells at the 1.06/1.07 g/mL interface were harvested and
washed twice in cold 0.15 mol/L NaCI. After viability count, the
cells were resuspended in PBS with 5% FCS and antibiotics ( IO7
cells/mL).
Labeling of cells with MoAbs andjlow cytometry. Flow cytometric analysis and cell sorting were conducted with a presterilized
FACStar-Plus flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), equipped with an argon ion laser
(Spectra Physics, Mountain View, CA) operated at 488 nm and 300
mW. Green (fluorescein isothiocyanate [FITC]) and red (phycoerythrin [PE]) fluorescence was detected using 530/30-nm and 585/
40-nm bandpass filters, respectively. Spectral overlap between
FITC and PE signals was electronically compensated. Data acquisition and analysis were performed with the FACStar PLUS Research
software (Becton Dickinson). For the enrichment of Lin-,
Lin-WGA', Lin-Sca- 1+,and Lin-Sca- I+WGA' cells, low-density
BM cells ( 107/mL) were first incubated with a cocktail of lineagespecific rat MoAbs, specific for murine T lymphocytes (CD4, clone
GK1.5; CD8, clone 53.6.7.), B lymphocytes (B220, clone RA36B2), granulocytes (Gr-1, clone RB6-8C5; PharMingen, San Diego,
CA), and macrophages (Mac-1, clone MI/70; Boehringer Mannheim, Indianapolis, IN). After 35 minutes of incubation on ice, the
cells were washed twice (800g for 10 minutes), resuspended at the
same density, and incubated another 30 minutes with PE-conjugated F(ab'), goat antirat IgG (AMAC, Westbrook, ME). After
washing (2X) cells were filtered through 37-pm nylon mesh to remove clumps and resuspended at 3 X lo6cells/mL in PBS with 5%
FCS and antibiotics. After the cells were analyzed, gates were set for
multiparameter sorting. The cells were sorted at a rate of 1,000 to
2,000 cells per second on the basis of intermediate forward scatter
(to exclude small lymphocytes and debris), low side scatter (to exclude highly granular cells), and low levels or lack of PE fluorescence (lineage negative cells [Lin-1). Forward and side scatter signals were collected on linear scales, and fluorescence signals were
collected on logarithmic scales. After completion of the sort, the
purity and viability of cells was determined, and cells were resuspended at lo7 cells/mL. The cells were then incubated 35 minutes
either with WGA-FITC (0.4 pglmL; Polysciences, Wamngton,
PA), with rat anti-Sca-1 (clone E13-161.7), or separately with both.
Cells labeled with anti-Sca-1 were incubated for another 30 minutes with PE-conjugated F(ab'), goat antirat IgG and washed twice.
After the final wash, cells were filtered through 37-pm nylon mesh
to remove clumps and resuspended at 3 X 106/mL in PBS with 5%
FCS and antibiotics. The cells were then sorted on the basis of
intermediate forward scatter and low side scatter and high levels of
FITC fluorescence (Lin-WGA' cells), high levels of PE fluorescence (Lin-Sca- 1+ cells), or both (Lin-Sca-l+WGA+ cells). After
completion of the sort, cells were extensively washed and counted,
and their viability was determined.
For enrichment of WGA' cells, BM cells were separated by density centrifugation on a Percoll gradient and simultaneously labeled
with WGA-FITC, as described p r e v i o u ~ l y . " ~ 'For
~ ~ ' ~the enrich-
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Sca-l+ CELLS IN Ly-6" MICE
2675
ment of Sca-l+WGA+cells, low-densitycells were first labeled with
anti-Sca-1 and then sorted for intermediate forward scatter, low
side scatter, and high levels of PE fluorescence (Sca-1' cells).
Enriched Sca- I + cells were then labeled with WGA-FITC and Scal+WGA+cells were sorted as described above. Sca-lfWGA+ cells
were isolated in two steps because the proportion of low-density
cells that express WGA-binding molecules (mainly Lin' cells) far
exceeds the proportion of Sca- 1 cells. Under such circumstances,it
is difficult to compensate the spectral overlap between FITC and
PE signals and immediate sorting of Sca-l+WGA+cells would result in high enrichment of WGA+ lineage-positivecells.
CFU-S assay. The CFU-S content of unfractionated and sorted
BM cells from FVB/N and BALB/c mice was determined by CFUS assay.37Recipient FVB/N and BALB/c mice were exposed to 10
Gy of y irradiation from a 137Cssource at a dose rate of 1 Gy/min.
Irradiated recipients ( 10 per group) received transplants intravenously with graded numbers of low-density (PO'")
WGA+,
,
ScaI+WGA', Lin-, Lin-WGA+, Lin-Sca- I+, and Lin-Sca- l+WGA+
cells. Animals were maintained on sterilized rodent chow and
aqueous antibiotics. Eight and 12 days after transplantation the
mice were killed by cervical dislocation, and their spleens were removed and fixed in Bouin's solution (Sigma, St Louis, MO). The
macroscopically visible spleen colonies (day-8 and day- 12 CFU-S)
were counted, and enrichment factor was determined. Control
mice that had been irradiated but not transplanted were included
for each group. The level of endogenous colony formation in irradiated controls was zero.
Radioprotection assay and long-term reconstitution. Lethally
irradiated (10 Gy) FVB/N and BALB/c mice have received transplants with graded numbers of Sca-I+WGA+, Lin-WGA+,
Lin-Sca- I+,and Lin-Sca- I+WGA+cells and were kept on aqueous
antibiotics for 3 weeks after irradiation. Radioprotection was assessed by following the survival of recipient animals for 30 days,
whereas long-term reconstitution was assessed by following the survival of the recipients 3 and 6 months after transplantation.
LTR ability of sorted cells. LTR ability of sorted cells (FVB/N
mice) was analyzed by qualitative assay using the polymerase chain
reaction (PCR) technique to identify the transgene sequence in hematopoietic cells of recipients. Lethally irradiated (10 Gy) FVB/N
mice have received transplants with graded numbers of Scal+WGA+, Lin-WGA', Lin-Sca- 1+, and Lin-Sca-l+WGA+ transgenic cells and kept on aqueous antibiotics for 3 weeks after irradiation. Twelve to 16 weeks after transplantation of sorted transgenic
cells, recipients were killed, and hematopoietictissues (spleen, BM,
thymus, and axilary lymph nodes) were harvested. Genomic DNA
was prepared from these tissues, and 100 ng of DNA was subjected
to PCR analysis. Oligonucleotide primers (sense primer sequence
was 5'-GTGAAGGAACCTTTCTGTGGTG-Y; antisense primer
sequence was 5'-GTCCTTGGGGTCTTCTACCTTTCTC-3') amplified a 3 10-bp fragment of the SV40 large T sequence. PCRs were
duplexed with primers for genomic ornithine transcarbamylase
(OTC) to provide a 131-bp fragment as an internal positive control
(sense primer sequence was 5'-TTTTCCCCTCTCAATACATTCACTGTC-3'; an antisense primer sequencewas S-AATGAAAGTCTCACAGACACCGCTCG-3'). Each DNA sample was analyzed
2 to 3 times. Amplification was performed in a Perkin-Elmer/Cetus
DNA Thermal Cycler (Norwalk, CT) for 30 cycles (denaturation 30
seconds at 95"C, annealing 30 seconds at 60"C, and extension 30
seconds at 72°C) using 0.4 pmol/L of each primer, 2.5 mmol/L of
each dNTP, Promega Taq buffer, and AmpliTaq DNA polymerase
(Perkin-Elmer/Cetus).PCR products were visualizedby UV illumination of the ethidium bromide-stained 1% agarose gels (SeaKem;
FMC Bioproducts, Rockland, ME). To determine the sensitivity
threshold of the PCR assay and to make it partially quantitative, a
+
set of DNA samples prepared from the mixed BM cell populations
containing loo%, 75%, 50%, 25%, lo%, 5%, 1%,and 0% of transgenic cells was subjected to PCR as described above. The similar
assay was performed by mixing DNA prepared from transgenic and
normal BM cells, and subjecting DNA samples containing loo%,
75%, 50%, 25%, lo%, 5%, 1%,and 0% oftransgenic DNA to PCR.
Secondary CFU-S assay. On killing of primary recipients who
had received transplants with 100 Sca- I'WGA+, Lin-WGA',
Lin-Sca-l+, and Lin-Sca-I+WGA+ transgenic cells, part of their
BM was used for secondary CFU-S assay. Groups of lethally irradiated (10 Gy) FVB/N mice have received transplants with lo5 BM
cells from primary recipients of each sorted cell phenotype. Twelve
days posttransplant, 5 mice from each group were killed, and their
spleens were harvested. Macroscopic colonies were counted, and
those well-separated were individually dissected with the aid of a
binocular dissecting microscope. Genomic DNA was prepared
from each colony and subjected to PCR analysis for detection ofthe
transgene sequence as described. PCR was performed for 25 cycles
to minimize the number of false-positive colonies contaminated
with surrounding cells of donor origin. The level of endogenous
colony formation in irradiated controls was zero.
RESULTS
CFU-S content of enriched BM populations. BM cells
were first separated by equilibrium density centrifugation
on a discontinuous Percoll gradient. This initial separation
of low-density cells (PI" cells) selected about 13% of the
total cells and increased the CFU-S concentration more
than 6 times (Table I , data shown for FVB/N mice only).
The analysis of P'Ow cells on the cytometer showed high heterogeneity of this population. For this reason a subpopulation of cells with intermediate forward scatter and low side
scatter ("blast window") was gated (Fig 1A) and used for
further sorting and enrichment. The gated subpopulation
retained greater than 94% of the initial number of CFU-S
cells (data not shown) and represented about 75% of P'Ow
cells. Similar blast window was used for sorting of WGA+,
Sca- 1+WGA+,Lin-, Lin-WGA', Lin-Sca- 1+,and Lin-Scal+WGA+cells (Fig 1). The proportion of cells gated for sorting (Fig 1) was expressed as a percentage of cells within the
same sorting window (same light scatter properties [X axis]
but different levels of fluorescence signal [Y axis]).
Low-density cells expressing high levels of WGA-binding
molecules (about 10%ofgated P'Owcells) gave rise to 1CFUS out of 46 cells at day 12, with the CFU-S,2/CFU-S8ratio of
1.15 (Table 1). Approximately 4% to 5% of unseparated BM
cells (data not shown) and 8%to 9%of gated Pow
cells were
positive for the Sca-1 antigen (Fig lB), and about 30% of
gated Sca-l+ cells expressed intermediate to high levels of
WGA-binding molecules (Fig 1C). Sorted Sca- l+WGA+
cells gave rise to 1 CFU-SI2out of 26 cells, with the CFUS12/CFU-S8ratio of 1.21 (Table 1). Labeling of Plowcells
with a cocktail of lineage-specificrat MoAbs and sorting on
the basis of intermediate forward scatter, low side scatter,
and low levels or lack of PE fluorescence (Fig 1D) has resulted in almost 50-fold enrichment of CFU-S12cells (Table
1). Approximately 20% of gated pow
cells was Lin-. Both
Lin-WGA'"- and Lin-WGAhi-sortedpopulations (Figs 1E
and F) were highly (436- and 490-fold, respectively)
enriched for CFU-SI2cells, but Lin-WGALoW
cells contained
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2676
JURECIC, VAN, AND BELMONT
Table 1. CFU-SCell Content of Sorted Ph,
EM Population
% of Whole EM
Whole bone marrow
100
13
1
0.26
1.95
0.14
0.2
0.1
0.043
plow
P'""WGA+
P'""Sca-1 +WGA+
PWinP1""Lin-WGA'""
P'""Lin-WGAh'
P'""Lin-Sca-1'
P'""Lin6ca- 1'WGA'
WGA+, Sca-1 +WGA+, Lin-, Lin-WGA', Lin- Sca-1 +,and Lin-Sca-I +WGA+ Cells
Mean No. of CFU-S,
per io3Cells
0.1 1
* 0.03
0.70 ? 0.078
18.70 f 4.3
31.53 2 7.4
4.60 f 1.65
5 0 f 11.7
33.32 f 5.9
5.16 1.12
28.30 f 4.8
Mean No. of CFU-Slz
per 1 O3 Cells
Enrichment Factor
(CFU-SIJ
0.11 k 0 . 0 2
0.70 0.056
21.50 ? 3.2
38.34 f 6.3
5.40 ? 1.92
48?11.5
54 ? 8.4
4 7 . 1 2 f 8.2
67.57 f 9.4
6.4
195 (1:46)'
348 (1 :26)
49 (1:185)
436 (1:21)
490(1:18)
428 (1:21)
614(1:15)
+
1
Lethally irradiated (10 Gy) recipients (10 per group) were transplanted intravenously with graded numbers (2 x 10' to 2 X 10') of low density (PI"),
WGA+, Sca-l+WGA+.Lin-, Lin-WGA+, Lin-Sca-l', and Lin-Sca-l'WGA+ cells. Eight and twelve days after transplantation the mice were killed by
cervical dislocation; their spleens were removed, and macroscopically visible spleen colonies (day-8 and day-1 2 CFU-S) were counted (data shown for
FVB/N mice only). The results represent mean values SD from three experiments.
* One CFU-SlZ cell per 46 WGA+ cells
+
1.5 times more CFU-s, cells (Table 1). Lin-WGA'O" cells
represented approximately 7%, whereas Lin-WGAhi cells
represented about 10% of P'""Lin- cells. Lin-Sca- I + cells
represented about 5% of P'O"Lin- cells (Fig 1G) and were
also highly enriched (428-fold) for CFU-S,2, but contained
sixfold to IO-fold lower number of cm-s, cells than
Lin-WGA' cells (Table 1).
Approximately 45% of Lin-Sca-1' cells express high levels, whereas about 40% express low to intermediate levels of
WGA-binding glucosamine residues. Sorting of cells that
coexpress Sca-1 antigen and high to medium levels of
WGA-binding glucosamine residues (Lin-Sca- 1+WGA+)
(Fig IH) resulted in 6 14-fold enrichment of CFU-S,, cells,
although a significant number of CFu-sS cells was
coenriched as well (Table 1). Sorted Sca-l+WGA+,
Lin-WGA+, Lin-Sca- 1+, and Lin-Sca- I+WGA+ cells from
the BM of BALB/c mice displayed similar enrichment of
CFU-S, and CFU-S,, cells (data not shown). Morphologically, sorted Sca- l+WGA+, Lin-WGA', Lin-Sca- 1+, and
Lin-Sca- I+WGA+cells from both strains were small lymphocyte-like cells or medium-sized blast cells.
Radioprotection and long-term survival. Radioprotection and long-term survival was assessed by transplanting
Lin-WGA',
graded numbers of sorted Sca-I'WGA',
Lin-Sca-1+, and Lin-Sca- l+WGA+cells and following the
survival of lethally irradiated recipients for 30, 90, and 180
days after transplant (Table 2, data shown for FVB/N mice
only). Four weeks after transplantation of 100,200,and 500
sorted Sca-l'WGA' cells, 70% to 100% of recipients had
survived, but the percentage of surviving animals had declined to 50%to 80%after 3 months and to 40% to 60%after
6 months. One month after transplantation of 100 sorted
Lin-WGA', Lin-Sca- 1+, and Lin-Sca- l+WGA+cells, 90%
to 100% of recipients had survived. Three and 6 months
after transplant, percentage of recipients that survived in
each group had declined to 40% to 50%, with the biggest
decline observed in recipients of Lin-Sca- 1+ cells. With increasing numbers of transplanted cells (200 and 500 cells,
respectively) increasing numbers of recipients survived over
the period of 3 and 6 months posttransplant (Table 2).
Again, the lowest percentage of survival was among the recipients of Lin-Sca- 1+ cells. None of the 15 control animals
that had not received any cells survived beyond 3 weeks
after irradiation. Enriched Sca- lCWGA+, Lin-WGA',
Lin-Sca-l+, and Lin-Sca-l+WGA+ cells from the BM of
BALB/c mice displayed similar radioprotection and longterm reconstitution capacity (data not shown).
L T R ability of sorted cells. Twelve to sixteen weeks after
transplantation, recipients of sorted transgenic cells were
killed, and hematopoietic tissues (spleen, BM, thymus and
axilary lymph nodes) were harvested. Genomic DNA prepared from these tissues was subjected to PCR analysis to
identify the presence of transgenic cells in hematopoietic
tissues of recipients. Figure 2 shows PCR amplification of
the transgene sequence and internal control (OTC) from a
set of DNA samples prepared from the mixed BM cell populations containing loo%,75%, 50%,2596, lo%, 5%, I%, and
0%of transgenic cells. This experiment, aimed to determine
the sensitivity threshold of the PCR assay, has shown that
the transgene sequence can still be amplified from a DNA
sample prepared from the cell population containing only
1% of transgenic cells. The same result was obtained with
the mixture of DNA from transgenic and normal BM, containing loo%, 75%, 50%, 25%, IO%, 5%, 1%, and 0% of
transgenic DNA, respectively.
After transplant of 100 Sca- I+WGA+ cells, donor cells
were present in all analyzed hematopoietic tissues in 2 recipients (Fig 3); in other 2 recipients, donor cells were not detected in lymph node thymus and BM, whereas l recipient
was negative in all tissues. Two of three mice receiving
transplants with 200 and 500 Sca-l+WGA+cells had transgenic cells in all analyzed tissues (Table 3). Two of five recipients of Lin-WGA+ cells (100 cells/mouse) had donor cells
in all tissues (Fig 3), whereas 1 mouse was negative for donor cells in BM, and 2 mice were negative in lymph nodes
(Table 3). Practically all mice receiving transplants with 200
and 500 Lin-WGA+ cells had transgenic cells in tissues analysed, with the exception of l mouse that was negative in
lymph nodes. Of 5 mice receiving transplants with 100
Lin-Sca-I+ cells, 3 were positive for donor cells in all tissues
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Sca-l+ CELLS IN L y a MICE
(Fig 3), whereas 2 mice were negative for donor cells either
in BM or in lymph nodes; 2 mice that received 200 cells
were also negative for donor cells in BM and lymph nodes
(Table 3).
The majority of mice that received Lin-Sca- l+WGAf
cells had donor cells present in all tissues analysed (Fig 3),
with the exception of 1 mouse that was negative in lymph
nodes (Table 3). Figure 3 shows the PCR analysis of hematopoietic tissues from 4 recipients, reconstituted with 100 Sca1+WGA+,Lin-WGA', Lin-Sca-l', and Lin-Sca- l+WGAf
cells. DNA isolated from BM and spleen cells of FVB/N and
transgenic mice served as the negative and positive control
for each PCR reaction. According to the sensitivity threshold determined for the PCR assay used herein (Fig 2), all
examined tissues regarded as positive contained at least 5%
of donor (transgenic) cells.
Secondary CFU-S assay. Groups of lethally irradiated
( 10 Gy) FVB/N mice have received transplants with IO5 BM
cells from primary recipients, which received transplants 16
weeks earlier with 100 Sca- l+WGAf, Lin-WGA', Lin-Sca1+, and Lin-Sca- l+WGA+ transgenic cells. Twelve days
posttransplant, 5 mice from each group were killed, and
their spleens were harvested. Well-separated macroscopic
colonies were individually dissected, and genomic DNA
was prepared from each colony and subjected to PCR analysis for detection of the transgene sequence as described.
PCR was performed for 25 cycles to minimize the number
of false-positive colonies contaminated with surrounding
cells of donor origin. The level of endogenous colony formation in irradiated controls was zero. After transplantation of
BM from primary recipients of 100 Sca- l+WGA+,
Lin-WGA', Lin-Sca- 1+, and Lin-Sca- l+WGA+ cells,
79.1%, 84.3%, 72.3%, and 83.6% of spleen colonies (CFUSI,) analyzed were positive for the transgene sequence (Table 4).
DISCUSSION
Cloning of the 12- to 18-kD glycosylphophatidylinositol
(GP1)-anchored Ly-6 proteins has shown that these molecules are encoded as a multigene family on mouse chromosome 15. One well-Characterized Ly-6 product is Ly-6A.2/
Ly-6E. 1 (Ly-6A/E), also referred to as T-cell-activating
protein and stem cell antigen 1 (Sca-1). Ly-6A.2 and Ly6E.1 molecules differ by only 2 of 108 amino acids and
appear to be products of allelic genes representing the Ly-6"
and Ly-sb haplotypes. Ly-6 molecules are expressed on subpopulations of T cells, thymocytes, B cells, BM cells, and
cells from kidney, liver, lung, heart, and brain. The function
of these molecules in lymphocytes remains unclear, although the binding of MoAb specific for Ly-6A/E regulates
both T- and B-cell function. In T cells anti-Ly-6A/E antibody initiates proliferation and increases interleukin-2 (IL2) secretion, whereas in B cells it regulates response to IL-4
The constitutive expresand interferon-y (IFN-y).3'-34,38,39
sion of Ly-BA/E on subsets of T cells, thymocytes, and B
cells was found on a substantially greater percentage of cells
obtained from C57BL/6 ( L Y - ~mice
~ ) than on those from
BALB/c (Ly-6") mice. However, high levels of Ly-6A/E are
2677
found on virtually all T and B cells after mitogen stimulation, regardless of the haplotype. Further studies have suggested that the haplotype-related difference in Ly-6A/E expression originates in differential regulation of Ly-6A/E
expression during lymphocyte development in fetal and neonatal thymus and spleen. Based on these observations, it
was suggested that BM cells from Ly-6" mice must be induced to express the Ly-6E. 1 molecule and, thus, cannot be
used for enrichment of stem cells expressing Sca-1 antigen.23,24
Approximately 4% and 7% of BM cells in L Y -and
~ ~L Y - ~ ~
mice express Ly-6A/E antigen, recognized by Sca- 1 antibody D7.33We have observed that about 4%to 5% ofunseparated BM cells and 8% to 9% of low-density BM cells from
FVB/N and BALB/c (Ly-6") mice show high to intermediate expression of Ly-6E.l antigen, as recognized by the
Sca- 1 antibody E 13-16 1.7.'9320To determine whether Ly6E. 1' cells from Ly-6" mice bear the same functional characteristics as Ly-6A.2' cells from L Y -mice,
~ ~ we have first
isolated Sca- l+WGA+and Lin-Sca- 1' cells and determined
their CFU-S content, radioprotection capacity, and ability
to reconstitute mice. Almost twofold higher CFU-SI2 cell
content in Sca- 1'WGA' population than in WGA+ cells
indicates that the initially isolated Sca- 1' cells contained
CFU-S cells. Long-term reconstitution and secondary CFUS assays have shown that the population of Sca-lfWGAf
cells from the BM of FVB/N mice includes primitive cells
with LTR capacity. Enriched Lin-Sca-l+ cells showed
CFU-SI, cell content, radioprotection, and LTR capacity
similar to Lin-Sca- 1' cells isolated from L Y - ~ ~
Altogether, these data show that, at least in FVB/N mice, cells
that constitutively express Ly-6E. 1 form of Sca- 1 antigen
contain CFU-S cells and cells with LTR capacity.
The lectin WGA has been useful for stem cell enrichments, due to a relatively high number of WGA-binding
sites on multipotent stem cells compared with lineage-committed progenitors. However, in none of the reports on
enriched WGA-binding cells have long-term studies been
reported on the ability of these cells to repopulate lethally
irradiated recipients.8,9,1 1,12,14,15, '7,' 8,4043 we have isolated
Lin-WGA' cells and determined their CFU-S content, radioprotection capacity, and ability to reconstitute mice. A
reference population of WGA' low density cells was almost
identically enriched for CFU-SI, cells (1 CFU-SI2cell out of
46 sorted cells) as in previous
Lin-WGA+ cells
were separated into two populations regarding the expression of WGA-binding sites. Although Lin-WGA'"" and
Lin-WGAhi cells were similarly enriched for CFU-SI, cells
(1 of 2 1 and 1 of 18 cells, respectively), Lin-WGA'"" cells
had a CFU-S,,/CFU-S, ratio of 0.96, whereas Lin-WGAhi
cells had a ratio of 1.62. This result confirms previous findings that CFU-S8 cells show lower affinity for WGA than
CFU-SI, cell^.'^,'^,'^ The CFU-S8/CFU-SI, cell content of
both populations was more than twofold higher than in
WGA' low density cells. Although similarly enriched for
CFU-SI, cells, Lin-WGA+ cells contained sixfold to 10-fold
higher number of CFU-S8 cells than did Lin-Sca-l+ cells
isolated from Ly-6" and L Y -mice.23
~ ~ In a qualitative LTR
assay, 100 Lin-WGA+ cells, isolated from transgenic FVB/
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2678
JURECIC. VAN, AND BELMONT
Fig 1. FACS profiles and window selection for sorting of Sca-1+WGA+, Lin-, Lin-WGA', tin-Sca-1 +,and Lin-Sca-1 +WGA+ cells
from the BM of FVB/N mice. Forward versus side light scatter of ?cells and selection of "blast" cells for further sorting (A). Profile of?
cells after labeling with Sca-1 and window selected for sorting of Sca-1+ cells (B). Profile of sorted Sca-1+ cells after labeling with
WGA-FITC and window selected for sorting of Sca-1+WGA+ cells (C).Profile of Ph cells after labeling with a cocktail of lineage-specific
MoAbs and window selected for sorting of tin- cells (D).
N (Ly-6") mice, have repopulated all hematopoietic tissues
in 40%of recipients, whereas 200 and 500 transplanted cells
have repopulated all tissues in 80% to 100%of recipients.
These results and the results of the secondary CFU-S assay
indicate that at least a part of primitive hematopoietic cells
with LTR capacity (LTR cells)bears the Lin-WGA+ phenotype.
Spangrude and S ~ o l l a yreported
~~
that Lin-Sca- I f cells
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2679
Sca-I+ CELLS IN Ly-6" MICE
Fig 1. (Cont'd). Profile of Lin- cells after labeling with WGA-FITC and windows selected for sorting of Lin-WGAh (E)and Lin-WGAh'
cells (F). Profile of Lin- cells after labeling with Sca-I and window selected for sorting of Lin-Sca-I cells (G). Profile of Lin- cells after
labeling with Sca-I and WGA-FITC and window selected for sorting of Lin-Sca-1 +WGA+ cells (H).
+
express high levels of carbohydrate binding sites for the lectin WGA but the expression of WGA-binding molecules in
relation to Sca- 1 antigen expression on cells with LTR ability has never been analyzed. Enriched Lin-Sca- l+WGA+
cells showed higher CFU-S,, cell content than did
Lin-WGA' and Lin-Sca-l+ cells but levels similar to those
found for Thy-1''Lin-Sca- 1+ cells. However, this population contained threefold to fivefold higher number of CFUS8 cells than did Thy- 1'"Lin-Sca- 1+ and Lin-Sca- I +
cells.'9*23,27,30
Radioprotection, long-term reconstitution,
and secondary CFU-S assays have shown that Lin-Sca-l+
cells that express high levels of WGA-binding glucosamine
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2680
JURECIC, VAN, AND BELMONT
Table 2. Radioprotection and Long-Term Survival of Primary
Recipients Receiving Transplants With Limiting Numbers of
Sca-1 +WGA+, Lin-WGA+, Lin-Sca-1 +,
and
Lin-Sca-1 +WGA+ Cells
EM
Population
Sca-1'WGA'
Lin-WGA'
Lin-Sca-1
+
Lin-Sca-l'WGA+
No. of
30-d
Cells
Injected
100
200
500
100
200
500
100
200
500
100
200
500
Survival
%
Survival
After 3
mo %
Survival
After 6
mo %
70 (7/10)
80 (8/10)
100(5/5)
100 (10/10)
100 (10/10)
100 (5/5)
90 (9/10)
100 (10/10)
100 (5/5)
90 (9/10)
100 (10/10)
100 (5/5)
50 (5/10)
60 (6/10)
80(4/5)
80 (8/10)
90 (9/10)
100 (5/5)
60 (6/10)
80 (8/10)
100 (5/5)
70 (7/10)
80 (8/10)
100 (5/5)
40 (4/10)
40 (4/10)
60(3/5)
50 (5/10)
70 (7/10)
80 (4/5)
40 (4/10)
50 (5/10)
60 (3/5)
50 (5/10)
70 (7/10)
80 (4/5)
Lethally irradiated (10 Gy) FVB/N mice have received transplants with
graded numbers of Sca-1'WGA', Lin-WGA', Lin-Sca-1+, and Lin-Sca1'WGA' cells. Radioprotectionwas assessed by following the survival
of recipient animals for 30 days, whereas long-term reconstitution was
assessed by following the survival of the recipients 3 and 6 months after
transplantation. Number of animals surviving/number of animals receiving transplants in parenthesis.
residues were highly enriched for LTR cells as well. Functional properties of Lin-Sca-I'WGA' cells suggest that, at
least in FVB/N (Ly-6') mice, the majority of CFU-SI, and
LTR cells coexpress the Ly-6E. 1 form of Sca-l antigen and
WGA-binding molecules.
In comparison with Lin-Sca-l+ cells, higher survival rate
of animals receiving transplants with 100 to 500
Lin-WGA' and Thy-1'OLin-Sca-l ' cells could be because
of the higher content of CFU-S8 cells that can generate a
sufficient number of committed progenitors and mature
cells necessary to prevent hematopoietic failure and to establish homeostatic hematopoiesis. A failure of IO0 transplanted Lin-WGA'. Lin-Sca- I+, and Lin-Sca- I 'WGA'
W
a
0
z
E
z
1;
-----
131 bp
Fig 2. PCR amplification of the transgene sequence (310 bp)
and internal control (ornithine transcarbamylase, 131 bp) from a
set of DNA samples prepared from the mixed EM cell populations
containing 100%. 75%. 50%. 25%. 10%.5%. 1%, and 0%of transgenic cells. This experiment, aimed to determine the sensitivity
threshold of the PCR assay. has shown that the transgene sequence can still be amplified from a DNA sample preparedfrom the
cell population containing only 1%of transgenic cells. $X174/
Haelll-digested molecular weight markers (MWM).
+
A
---- 3 10 bp
d
B
C
D
I ---- 131 bp
L
E
Fig 3. PCR analysis of DNA prepared from hematopoietic tissues of recipients reconstituted with 100 Sca-1+WGA+ (A),
Lin-WGA+ (E), Lin-Sca-1 (C), and Lin-Sca-1 +WGA+ (D)
transgenic cells. A 310-bp fragment amplified from the transgene was
detected in EM, spleen, thymus, and lymph node of recipients 12to
16 weeks after transplant. All PCR reactions were duplexed with
primers for genomic OTC to provide a 131-bp fragment as an internal positive control. Negative control ( - ) was DNA prepared from
EM cells of FVB/N mice; positive control [ ), DNA prepared from
EM cells of transgenic FVB/N mice; $X174/Haelll-digested rnolecular weight markers (MWM). Genomic DNA (100 ng) was amplified
as described.
+
+
cells to repopulate all hematopoietic tissues in 20% to 60%
of recipients could be due to a suboptimal number of LTR
cells transplanted, a low level of their long-term engraftment, or a loss of their repopulating capacity. Furthermore,
long-term survival and reconstitution of recipients of the
small graft could be largely mediated by the host stem cells
that had survived i r r a d i a t i ~ n . ' ~as. ~was
~.~
shown
~
in animals
and 30 to 100
that received 20 and 33 Lin-Sca-1'
Thy-1'OLin-Sca- I cells.30Quantitative LTR assays using
different Ly-5 allelic locus have shown that only animals that received greater than 500 Lin-Sca-I' or Thy1'OLin-Sca-I' cells have about 90% or more cells of donor
origin. Furthermore, long-term reconstitution of myeloid
and lymphoid compartment by donor cells is mostly observed in recipients transplanted with higher numbers of
enriched Is." IO. 16.22.23.27.19.30
Although the exact content of donor cells in hematopoietic tissues of recipients transplanted with Sca-I+WGA',
Lin-Sca-1'. Lin-WGA', and Lin-Sca- I'WGA' cells could
not be determined, based on the sensitivity threshold ofthe
+
----- 310 bp
I
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2681
Sca-l+ CELLS IN Ly-6' MICE
PCR assay it was estimated that all examined tissues regarded as positive contained at least 5% of donor (transgenic) cells. However, the high percentage of donor-derived
spleen colonies in secondary recipients shows that the content of donor cells in primary recipients is in agreement with
previous studies that have shown that 8 to 12 weeks after
transplantation of 100 Lin-Sca- 1' or Thy- 1'"Lin-Sca- 1+
cells approximately 30% to 75%of peripheral blood leukocytes are of the donor origin.'0*23s30
In brief, functional analysis of enriched Sca- l+WGA+,
Lin-Sca- I+, Lin-WGA', and Lin-Sa- l+WGA+ BM cells
from two mouse strains with Ly-6' haplotype has shown
that (1) CFU-S cells and cells with LTR capacity constitutively express the Ly-6E. 1 form of Sca-1 antigen, and that (2)
the hematopoietic stem cell activity in FVB/N mice, defined by LTR of hematopoietic tissues in irradiated animals, is correlated with the expression of Ly-6A/E antigen
and WGA-binding molecules. Furthermore, the results suggest that Sca-1 antigen expression in stem cells is not restricted to mice with a particular genetic background, but
rather could be an invariant characteristicof mouse hematopoietic stem cells. This latter point underscoresthe probable
functional importance of Ly6A/E molecule in the biology
of hematopoietic stem cells."~"
The mouse Ly-6 antigens are members of the Ly-6 superfamily of GPI-anchored molecules that include squid SGp2
gene of unknown function and human CD59 cell surface
antigen (mouse Ly-6 homologue) and urokinase plasminogen activator receptor. Human CD59 glycoprotein regulates
the formation of the polymeric C9 complex of complement
and is deficient in the hematopoietic cells of patients with
Table 3. LTR Ability of Sca-I +WGA+. Lin-WGA',
and tin-Sca-1 +WGA+ Cells
BM
Population
Sca 1+WGA+
Lln-WGA+
Lin-Sca- 1+
Lin-Sca-l+WGA+
No. of Cells
Injected
(no. of
animals
analyzed)
Lln-Sca-l+,
No. of Animals With Transgenic Cells in
BM
Spleen
Thymus
Lymph Node
3
2
4
3
2
2
3
3
3
2
3
2
200 (3)
500 (3)
4
3
3
3
3
5
3
3
3
2
3
5
3
3
5
3
3
2
5
5
3
3
lOO(5)
200 (3)
500 (3)
lOO(5)
lOO(5)
4
200 (3)
500 (3)
2
100 (5)
200 (3)
500 (3)
5
3
3
3
5
3
3
4
3
4
3
3
LTR ability of sorted cells was analyzed by qualitative assay using the
PCR technique to identify the transgene sequence in hematopoietic cells
of recipients. Twelve to 16 weeks after transplantation of sorted transgenic cells, recipients were killed and hematopoietic tissues (spleen,
BM, thymus, and axilary lymph nodes) were harvested. Genomic DNA
was prepared from these tissues, and 100 ng of DNA was subjected to
PCR analysis.
Table 4. Origin of Secondary Spleen Colonies Generated by
Transplantation of BM From Primary Recipients Reconstituted
With 100 Sca-1 +WGA+, tin-WGA+, Lln-Sca-1 +,and
Lin-Sca-1 +WGA+ Transgenic Cells
~
BM
Population
% of Secondary Spleen Colonies
Positive for Transgene Sequence
Sca- 1+WGA+
Lln-WGA+
Lin-Sca-I +
Lin-Sca-I +WGA+
79.1 (19/24)'
84.3 (27/32)
72.3 (34147)
83.67 (41149)
On killing of primary recipients receiving transplants with 100 Sca1+WGA+, Lin-WGA+, Lin-Sca-I +,and Lin-Sca- 1+WGA+ transgenic
cells, part of their BM was used for secondary CFU-S assay. Groups of
lethally irradiated (10 Gy) FVB/N mice have received transplants with
lo5 BM cells from primary recipients of each sorted cell phenotype.
Twelve days posttransplant, 5 mice from each group were killed; their
spleens were harvested, and well-separated colonies were dissected.
Genomic DNA were prepared from each colony and subjected to PCR
analysis for detection of the transgene sequence.
Number of positive colonies/number of colonies analyzed in parenthesis.
paroxysmal nocturnal hemoglobin~ria.~"~~
It is well established that Ly-6 antigens play a regulatory role in B- and
T-cell activation. As GPI anchored molecules that are differentially expressed in primitive and mature hematopoietic
cells, Ly-6 antigens (along with Thy-1 antigen) could be
involved in the regulatory pathway affectingthe very early
stage of stem cell ontogeny. Their expression in T cells, B
cells, thymocytes, and BM cells seems to be regulated by
IF"--, and tumor necrosis factor (TNF).31-33,39
Recent experiments have established that both cytokines inhibit differentiation of murine BM progenitors (Lin- cells), where
TNF inhibits their differentiation in part through downmodulation of colony-stimulating factor receptor expresion.^^^^' Furthermore, IFN-7 has been shown to inhibit
myelopoiesis and lymphopoiesis in long-term BM cultures
and to upregulate expression of Ly-6 molecules in several
B-cell lines but not in stromal cells.s2Because both IFN-7
and TNF can upregulate Ly-6 expression in hematopoietic
cells and inhibit differentiation of murine progenitor cells, it
is tempting to speculate that IFN/TNF-mediated modulation of Ly-6 expression could be involved in the regulation
of hematopoietic stem-cell proliferation and/or differentiation. The finding that a somatically acquired loss of CD59
expression is associated with clonal outgrowth and domin a n ~ is
e ~consistent
~
with the possibility that Ly-6 molecules participate in growth regulation within the early precursor compartment. However, a thorough analysis of
Lyd-expression modulation and ligand identification are
necessary to elucidate the function of Ly-6 molecules in
hematopoietic stem cells.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 82: 2673-2683
Enrichment and functional characterization of Sca-1+WGA+,
Lin-WGA+, Lin- Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from
mice with an Ly-6a haplotype
R Jurecic, NT Van and JW Belmont
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