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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-06-1918.
HEMATOPOIESIS
From the Programme in Cancer/Blood, Hospital for Sick
Children, Toronto, ON, Canada; the Programme in Development and Fetal
Health and the Programme in Molecular Biology and Cancer, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON,
Canada; and the Department of Molecular and Medical
Genetics and the Institute of Biomaterials and Biomedical Engineering,
University of Toronto, ON, Canada.
Despite its wide use as a marker for hematopoietic stem cells
(HSCs), the function of stem cell antigen-1 (Sca-1) (also
known as lymphocyte activation protein-6A
[Ly-6A]) in hematopoiesis remains poorly defined. We
have previously established that Sca-1 Hematopoiesis proceeds through the differentiation
of stem cells into a cascade of committed progenitors and, finally,
into all of the terminally differentiated cell lineages of the
blood system. At least in C57BL/6 and other Ly-6.2
mouse strains, the cell surface phenotype of the hematopoietic stem
cells (HSCs) and primitive progenitors has been well
elucidated. One of the markers whose expression defines the HSCs and
various developmental intermediates is the glycosyl
phosphatidylinositol-anchored protein (GPI-AP) stem cell antigen-1
(Sca-1). Single lineage-negative (Lin All functional analysis of Sca-1 has been performed in primary
lymphocytes or lymphoid cell lines. The Sca-1 protein (or lymphocyte activation protein-6A [Ly-6A]) was originally identified as an antigen up-regulated on activated lymphocytes.8 GPI-APs
are believed to facilitate cell signaling by creating glycosphingolipid cholesterol "rafts" that concentrate or exclude specific signaling molecules.9,10 Consistent with this mechanism, Src family kinase members have been shown to associate with Sca-1.11
Several members of the Ly-6 gene family, which share similar
structural motifs as well as primary sequence, have identified cognate
ligands, such as the Ly-6 protein CD59, which protects cells from
complement-mediated lysis by binding the The absence of an identified ligand has made determination of the role
of Sca-1 in cell signaling difficult. Simulation of ligand binding by
cross-linking Sca-1 with anti-Sca-1 antibodies has generated
conflicting results.15-18 However, antibody cross-linking assays are artificial and probably do not represent physiologic ligand-receptor interactions. Therefore, we undertook a genetic approach to determine the function of Sca-1.19 Homologous
recombination was used to ablate the Sca-1 gene in 129 ES
cells. Homozygous mutant mice were produced at normal Mendelian
frequencies, demonstrating that Sca-1 is not required for development.
However, T cells from Sca-1-deficient mice demonstrate significantly
higher and more prolonged proliferation in response to
stimulation through the T-cell-receptor complex as compared with T
cells from Sca-1+/+ littermates. Although the
mechanism driving hyperproliferation has not been determined, increased
cytokine production, skewed thymocyte development, and overall
expression of Src family kinases have been ruled out (Stanford et
al,19 and W.L.S., unpublished observations,
1998). Consistent with these results, overexpression of Sca-1 in T
cells inhibits CD4+ T-cell proliferation induced by
T-cell-receptor activation.20 Together, these results
suggest that Sca-1 acts to down-modulate an active immune response.
Furthermore, we have recently demonstrated that Sca-1 is required for
the immunosuppressive function of CD4 To analyze the developmental potential of Sca-1 Animals
Antibodies
Cytokines Purified thrombopoietin (recombinant human thrombopoietin [rhTPO]), rhIL-6, and rhIL-11 were purchased from PeproTech (Rocky Hill, NJ). Purified rhIL-7 was purchased from Stem Cell Technologies (Vancouver, BC, Canada), and recombinant mouse IL-3 (rmIL-3) was purchased from BD PharMingen.Flow cytometry Single cell suspensions were prepared from bone marrow (tibial and femoral). The bone marrow was subjected to red blood cell (RBC) lysis with ammonium chloride, washed once in Iscove Dulbecco modified Eagle medium (DMEM), counted, and resuspended in phosphate-buffered saline (PBS) plus 2% fetal bovine serum (FBS) (staining medium [SM]) at 5 × 106 cells per milliliter. Then, 106 cells were incubated with mAbs for 30 minutes, washed twice in SM, and resuspended in 0.2 mL SM for analysis by means of a Becton Dickinson FACScalibur (Mountain View, CA).Glucose phosphate isomerase 1 (Gpi1) isoenzyme analysis The samples were subjected to electrophoresis on cellulose acetate plates (Helena Laboratories, Beaumont, TX), and the Gpi1 isoenzyme bands were visualized as previously described.22 The contribution of mutant cells to the samples was visually estimated by comparing them with a set of standard mixtures of Gpi1-AA and Gpi1-BB cells.Hematologic analysis Peripheral blood (50 to 100 µL) from tail bleeds was collected into EDTA (ethylenediaminetetraacetic acid)-coated capillary pipettes (Drummond, Broomall, PA) and transferred to Eppendorf (Hamburg, Germany) tubes. Complete blood counts and differential counts were performed by means of a Coulter Ac-T Differential Hematology Analyzer with veterinary software (Beckman-Coulter, Fullerton, CA).Bleeding time assay Six- to 8-week-old mice were anesthetized with the use of 0.15 mg ketamine per gram body weight to reduce influences of heart rate and activity on bleeding time. Approximately a 3-mm portion of the tail was cleanly severed with the use of scissors. The tail was immersed in physiologic saline at 37°C. The time required for the small stream of blood to stop was recorded as the bleeding time.Culture CFUs For each experiment, 3 animals of each genotype were used. Each experiment was performed between 3 and 8 times. Single cell suspensions were prepared from bone marrow (tibial and femoral). Cells were counted and diluted to 106 cells per milliliter in Iscove modified Dulbecco medium (IMDM) with 1% FBS. For each assay, 3 aliquots of 104 cells were mixed with the appropriate methylcellulose media, plated, and grown in humidified chambers at 37°C, 5% CO2. To assay megakaryocyte CFU precursors (CFU-Mk), cells were grown in methylcellulose/collagen medium (Stem Cell Technologies) containing 50 ng/mL rhTPO, 10 ng/mL rmIL-3, 20 ng/mL rhIL-6, and 50 ng/mL rhIL-11, and visualized by acetylcholinesterase activity. All other CFU-C colonies were grown in methylcellulose containing IL-3, IL-6, Steel factor (SLF), and erythropoietin (EPO) (M3434; Stem Cell Technologies). Erythroid CFU (CFU-E) precursors were assayed after 2 days by staining in situ with benzidine (Sigma, St Louis, MO) to detect hemoglobin. After 7 to 10 days, erythroid burst-forming units (BFU-Es) and CFU-Cs were counted by colony morphology. Representative colonies were occasionally analyzed by Wright-Giemsa to confirm colony identity.Spleen CFUs The results shown are combined from 3 separate experiments. For each experiment, we combined the bone marrow from 3 mice of the same genotype. Single cell suspensions were prepared from bone marrow flushed from both tibiae and femora and diluted to 105 cells per milliliter. Then, 104 or 2 × 104 cells were injected into the tail vein of sublethally irradiated (8.0 Gy, 137C source) mice. For each experiment, 6 recipients were used for each donor genotype. Recipients were humanely killed on day 12 of transplantation, and their spleens were fixed with Bouin solution. Macroscopic colonies were counted. Spleens from irradiated mice that did not receive cells contained on average less than 1 CFU-S colony.Transplantation assays For each repopulation experiment, 6 C57BL/6J-Gpi1a/a and 6 C57BL/6J-Gpi1b/b (either Sca-1 / or Sca-1+/+
used in some control experiments) bone marrow donor mice were treated with 150 µg 5-fluorouracil (5-FU) (Sigma) per gram body weight. At 2 days later, bone marrow (tibial and femoral) was flushed from each donor, and the bone marrow cells were
combined according to Gpi1 haplotype. Whole bone marrow cells were then subjected to iso-osmotic Percoll (Pharmacia, Piscataway, NJ)
centrifugation. The density-separated (1.077 g/mL) donor bone marrow
cells (or "buffy coats") were then mixed at the following
ratios for competitive repopulation: 100%
Gpi1a/a to 0% Gpi1b/b,
67.7% Gpi1a/a to 33.3%
Gpi1b/b, 33.3% Gpi1a/a
to 67.7% Gpi1b/b, and 0%
Gpi1a/a to 100% Gpi1b/b.
Bone marrow recipients (12 for each experiment) were treated with a
single-dose, total body lethal irradiation (9.5 Gy, 137C
source). Then, 5 × 106 cells of each separated bone
marrow mixture (a total of 4 groups) were injected into the tail vein
of 3 recipients. Tail bleeds were performed at 2, 4, 8, and 16 weeks
for analysis of bone marrow contribution by Gpi1 chimerism. In some
experiments, blood samples were obtained at 32 and 52 weeks. No
significant differences were observed between 16 and 52 weeks. Bone
marrow was isolated from recipients at either 16 or 52 weeks, analyzed
for Gpi1 chimerism, and used for secondary repopulations. Bone marrow
was harvested from 2 groups of 16-week-long primary bone marrow
recipients for secondary transplantation: (1) wild-type
(Gpi1a/a) irradiated mice were repopulated with
Sca-1/ (Gpi1b/b) bone marrow;
and (2) Sca-1/ (Gpi1b/b)
irradiated mice were repopulated with wild-type
(Gpi1a/a) bone marrow. For each primary
recipient, 5 × 106 Percoll-separated bone marrow cells
were injected into the tail veins of 3 lethally irradiated mice of the
same genotype (and Gpi1 isotype) as the primary recipient.
Hematologic analysis To minimize genetic background effects on the analysis of the role of Sca-1 in hematopoiesis, we backcrossed the Sca-1-targeted null allele onto the C57BL/6 background for 10 generations. Theoretically, the entire genetic background of these animals is derived from the C57BL/6 strain with the exception of approximately 20 cM flanking the Ly-6 locus, which is derived from the 129 ES cell line used for gene targeting.19 The backcross-10 mice were bred to generate Sca-1+/+ and Sca-1/
progeny, which were subsequently independently bred. Hematopoietic analysis of Sca-1/ mice began with automated
peripheral blood counts (Table 1). The
hematocrits, hemoglobin levels, and total RBC and WBC counts were
similar in the Sca-1+/+ and
Sca-1/ mice. Yet the percentages in the
peripheral WBC counts were significantly altered in
Sca-1/ mice (P < .05). The
percentage of peripheral blood lymphocytes was increased, with a
concomitant decrease in both monocytes and granulocytes. These results
are consistent with our previous report that lymphocytes derived from
Sca-1/ mice are hyperproliferative in
response to antigen.19 Unexpectedly, peripheral blood
analysis revealed that Sca-1/ mice have 31%
fewer platelets (P < .01). Next, flow cytometric analysis
was performed to compare the number and percentages of bone marrow
lineages in Sca-1+/+ and
Sca-1/ mice. Bone marrow cellularity was
found to be normal in Sca-1/ mice as were
the proportions of monocytes, granulocytes, erythroblasts, and
lymphocytes; however, bone marrow from Sca-1/
mice exhibited a marked reduction in megakaryocytes (by greater than 75%), consistent with the decrease in peripheral blood platelets (Table 2; P < .01). In
addition, histologic examination of adult spleens also showed a
reduction in megakaryocytes (data not shown).
To determine if the defective megakaryopoiesis resulted in a
physiologic defect, bleeding times in response to tail cuts were measured on Sca-1+/+ and
Sca-1 Precursor analysis Colony-forming assays were performed to analyze the role of Sca-1 in the development of myeloid-committed precursors. The most primitive in vitro colony-forming cell (granulocyte, erythroid, macrophage, megakaryocyte CFU [CFU-GEMM]) was reduced by more than 60%. As expected by the 4-fold decrease in bone marrow megakaryocytes, there was also a substantial decrease in megakaryocyte precursors (megakaryocyte CFUs [CFU-Mk's]) in Sca-1/ bone marrow (Table
3). Consistent with unaltered percentages of RBCs and TER-119+ cells in the periphery and
bone marrow, Sca-1/ bone marrow contained
normal percentages of erythroid (both BFU-E and CFU-E) progenitors.
Although peripheral blood and bone marrow granulocyte and monocyte
numbers are modestly decreased in Sca-1/
mice, CFU-GM, CFU-M, and CFU-G precursors are all significantly increased in mutant marrow. The CFU-spleen day-12 (CFU-S12)
assay is an in vivo clonogenic method that measures a primitive
progenitor. As shown in Figure 1,
Sca-1/ bone marrow has a 38% decrease in
CFU-S12 progenitors.
Hematopoietic stem cell analysis Competitive bone marrow repopulations were performed to measure HSC activity in Sca-1/ mice. First, lethally
irradiated mice received 5 × 106 medium-density
Sca-1+/+ and Sca-1/
bone marrow cells that had been mixed at specific ratios.
Wild-type and mutant cells were distinguished by expression of the
glucose phosphate isomerase 1 (Gpi1) isoenzyme expressed by
all cells, including erythrocytes. Wild-type bone marrow cells (from
the C57BL/6J-Gpi1a/a strain) express the Gpi1AA
isoenzyme, whereas control Sca-1+/+ and
Sca-1/ bone marrow cells express the Gpi1BB
isoenzyme. When mixtures of wild-type Gpi1AA and Gpi1BB bone marrow
were used to repopulate lethally irradiated wild-type Gpi1AA mice,
approximately the same percentage of input donor cells was recovered
from the bone marrow of long-term repopulated animals (Figure
2, square dotted line), closely
resembling the expected values depicted in the solid line. In
contrast, when mixtures of wild-type Gpi1AA and
Sca-1/ bone marrow cells were used to
repopulate lethally irradiated Gpi1AA mice, a significantly greater
proportion of Gpi1AA cells were recovered from the bone marrow of
long-term repopulated mice (Figure 2, dashed line). For
example, mice receiving transplants of bone marrow at a ratio of 2:1
mutant-to-wild-type cells showed reconstitution by wild-type cells at
more than twice the expected frequency. In addition, Gpi1 analysis of
peripheral blood from mice that have received transplants suggested
that Sca-1/ bone marrow had impaired
short-term repopulating activity (data not shown). Yet no differences
in Gpi1 ratios were observed in 4-month and 12-month repopulated mice.
Surprisingly, the Gpi1AA mice repopulated with 100%
Sca-1/ Gpi1BB bone marrow (BM),
showed only an average of 80% Gpi1BB bone marrow. Thus, some
endogenous wild-type Gpi1AA HSCs recolonized the bone marrow despite
the transplantation of 5 × 106
Sca-1/ bone marrow cells.
Reciprocal experiments were conducted in which
Sca-1 Serial transplantations HSC self-renewal activity was analyzed by using serial transplantation of Sca-1/ bone marrow into
wild-type Gpi1AA mice and of wild-type Gpi1AA bone marrow into
Sca-1/ mice (Figure
3). As shown in Figures 2 and 3,
transplantation of 5 × 106 wild-type bone marrow cells
into lethally irradiated Sca-1/ mice
demonstrated complete reconstitution by wild-type cells. At 4 months
after transplantation, bone marrow was harvested for Gpi1
isoenzyme analysis, and the remaining cells were subjected to gradient
centrifugation. For each primary recipient, 5 × 106
medium-density bone marrow cells were injected into the tail veins of 3 lethally irradiated Sca-1/ mice. All of the
secondary recipients demonstrated complete reconstitution of wild-type
cells into Sca-1/ mice by 4 months after
transplantation. However, repopulation of wild-type mice with the use
of Sca-1/ bone marrow demonstrated
remarkably different results. As shown in Figures 2 and 3, primary
wild-type mice that received transplants of
Sca-1/ bone marrow exhibited poor donor
engraftment. Secondary transplants from these animals into lethally
irradiated wild-type mice resulted in about 45% engraftment by
Sca-1/ bone marrow in the 6 recipients that
survived transplantation. Three of the 9 secondary transplant
recipients died within 17 days after transplantation. The moribund
animals demonstrated hematocrits of less than 20% measured a week
after transplantation.
Loss of Sca-1 and diminished Kit signaling reduces embryonic survival The receptor tyrosine kinase c-Kit encoded by the white-spotted (W) locus23 is required for hematopoietic development and HSC self-renewal. The white-spotted viable (Wv) allele of c-Kit is a homozygous viable mutation that results in a threonine-to-methionine substitution at position 660 in the kinase domain resulting in reduced kinase activity.24 To assess the functional capabilities of Sca-1/ HSCs in situ (ie, without ex vivo
manipulation and transplantation), Sca-1/
mice were bred with Wv/+ mice to
generate Sca-1/;Wv/+ mutants,
which were then intercrossed. While
Wv/+ intercrosses generated 22%
Wv/Wv homozygous pups,
which exhibit mild anemia and impaired HSC competition, CFU-S activity,
and decreased CFU-C precursors,
Sca-1/;Wv/+ intercrosses
generated only 5%
Sca-1/;Wv/Wv
compound homozygote pups, indicating that most
Sca-1/;Wv/Wv
compound homozygotes die in utero (P < .0001; Figure
4). Preliminary analysis of embryonic day
-14 litters from Sca-1/;Wv/+
intercrosses indicate that compound homozygotes are very anemic and
have dramatically reduced CFU-Cs, suggesting that the cause of
embryonic lethality is due to a stem cell or very early progenitor defect (C.Y.J.L., N. Ciliberti, and W.L.S., unpublished
data, 2002).
The data presented here demonstrate that Sca-1 plays an important
role in regulating the repopulating ability of HSCs and the development
of committed progenitor cells, megakaryocytes, and platelets. The
decrease in CFU-S and CFU-GEMM progenitors may be attributable to
deficient stem cell self-renewal and differentiation observed by means
of the repopulation assays. The simplest explanation for the dramatic
reduction in Sca-1 Under homeostatic conditions (ie, Sca-1-null mice), Sca-1 is not
required for stem cell maintenance, which is consistent with a role for
Sca-1 as a coregulator of cell signaling; in this case, however, a coregulator of the signals that mediate stem cell
self-renewal. A postulated molecular role of Sca-1 and other
GPI-anchored proteins is to modulate signaling by Src family kinases,
receptor tyrosine kinases, and other signaling molecules in lipid
rafts. Sca-1 and the receptor tyrosine kinase c-Kit are coexpressed on
HSCs, CLPs, early myeloid progenitors, and mast cells.1,3
While null Kit mutations lead to embryonic lethality owing
to severe anemia caused by decreased stem cells and progenitors, the
partial loss of function-W alleles, such as
Wv/Wv and
W41/W41, has impaired HSC
competition and CFU-S activity, decreased CFU-Cs, and lead to
mild anemia, mast cell deficiencies, and mild thrombocytopenia.28,29 Thus, the HSC and myeloid
progenitor phenotype of Sca-1 In addition to biochemical association of Sca-1 and Src family
kinases,11 there is genetic evidence that Sca-1 and other GPI-APs are negative regulators of Src family kinases. For example, the
lymphoid phenotypes of Sca-1 and Src family kinase gene-targeted mice
are diametrically opposite; Sca-1 Significantly, our finding that Sca-1 plays a functional role in HSC
self-renewal is consistent with the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), an acquired disease resulting from a
clonal expansion of HSCs that harbor a somatic null mutation in the
X-linked phosphatidylinositol glycan-class A (Pig-A) gene, which catalyzes the first step in GPI biosynthesis.33 PNH
is characterized by complement-mediated intravascular hemolysis and eventual bone marrow failure. Gene-targeting experiments have shown
that the lack of the GPI-anchored complement inhibitors CD55
(decay-accelerating factor [DAF]) and CD59 are the primary cause for the complement-mediated hemolysis affecting PNH
patients.34,35 However, the mechanism enabling the PNH
stem cell to expand within the bone marrow is not due to a loss of
GPI-APs. GPI-deficient clones encoding PIG-A mutations have
been detected in healthy individuals in trace amounts without the
tendency to expand.36 Furthermore, mice chimeric for a
germ line-targeted mutation in Pig-A actually lose
Pig-A In addition to HSCs, Sca-1 expression has been reported on muscle,
bone, and mammary gland stem and/or primitive
progenitors.38-41 Consistent with the function of Sca-1 in
HSCs, we have recently found that Sca-1
The authors would like to thank Dwayne Barber, Jason Cohn, Norman Iscoves, Tammy Reid, and Peter Zandstra for critical reading and helpful discussions. This work is dedicated to the memory of Ms Karyn Glick.
Submitted June 20, 2002; accepted August 14, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-06-1918.
Supported by grants from the Canadian Institutes of Health Research, Ottawa, ON, Canada; the Leukemia and Lymphoma Society, New York, NY; and the Terry Fox Foundation, Vancouver, BC, Canada. C.Y.I. was supported by a National Research Service Award (NRSA) training fellowship (National Institutes of Health [NIH]); and W.L.S. is the Karyn Glick Memorial Special Fellow of the Leukemia and Lymphoma Society of America and the Canadian Research Chair in Stem Cell Biology and Functional Genomics.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William L. Stanford, Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Rd, Rm 407 Rosebrugh Bldg, Toronto, ON M5S 3G9 Canada; e-mail: william.stanford{at}utoronto.ca.
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Top
Abstract
Introduction
-chain of complement 8 and
the "b" domain of complement 9 (Ninomiya and Sims
,
anti-macrophage adhesion molecule-1 (anti-MAC-1) (CD11b),
anti-CD41 (integrin 
3), anti-Thy-1, anti-Kit, anti-IL-7R
] versus 3.2 CFU-S/104 Sca-1
];
P < .01) in CFU-S12 compared with wild-type
bone marrow. The results are shown as the percentage of colonies from
wild-type bone marrow from 3 separate experiments.
