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Blood, 15 August 2000, Vol. 96, No. 4, pp. 1380-1387
HEMATOPOIESIS
Homing and engraftment potential of
Sca-1+lin cells fractionated on the basis of
adhesion molecule expression and position in cell cycle
Christie M. Orschell-Traycoff,
Kelly Hiatt,
Ramzi N. Dagher,
Susan Rice,
Mervin C. Yoder, and
Edward F. Srour
From the Division of Hematology/Oncology and Indiana
Elks Cancer Research Center, Department of Medicine; Herman B Wells
Center for Pediatric Research, Department of Pediatrics; and Department
of Microbiology and Immunology, Indiana University School of Medicine,
Indianapolis.
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Abstract |
Engraftment potential of hematopoietic stem cells (HSCs) is likely
to be dependent on several factors including expression of
certain adhesion molecules (AMs) and degree of mitotic quiescence. The
authors investigated the functional properties and engraftment potential of Sca-1+lin cells subfractionated
on the basis of expression, or lack thereof, of CD11a, CD43, CD49d,
CD49e, or CD62L and correlated that expression with cell cycle
status and proliferative potential of engrafting fractions.
Donor-derived chimerism in mice receiving CD49e+ or
CD43+ Sca-1+lin cells was greater
than that in mice receiving cells lacking these 2 markers, while
Sca-1+lin cells positive for CD11a and CD62L
and bright for CD49d expression mediated minimal engraftment. AM
phenotypes enriched for engraftment potential contained the majority of
high proliferative potential-colony forming cells, low
proliferative potential-colony forming cells, and cells providing
rapid in vitro expansion. Cell cycle analysis of AM subpopulations
revealed that, regardless of their bone marrow repopulating potential,
Sca-1+lin AM cells contained a
higher percentage of cells in G0/G1 than their AM+ counterparts. Interestingly, engrafting phenotypes,
regardless of the status of their AM expression, were quicker to exit
G0/G1 following in vitro cytokine stimulation
than their opposing phenotypes. When engrafting phenotypes of
Sca-1+lin AM+ or AM
cells were further fractionated by Hoechst 33342 into
G0/G1 or S/G2+M, cells providing
long-term engraftment were predominantly contained within the quiescent
fraction. These results define a theoretical phenotype of a
Sca-1+lin engrafting cell as one that is
mitotically quiescent, CD43+, CD49e+,
CD11a, CD49ddim, and CD62L.
Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs.
(Blood. 2000;96:1380-1387)
© 2000 by The American Society of Hematology.
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Introduction |
The ability of hematopoietic stem cells (HSCs) to
reconstitute normal bone marrow (BM) hematopoiesis following
transplantation into suitable recipients relies on the potential of
these cells to home to and anchor within the BM microenvironment.
Homing is an intricate process by which HSCs, through interactions
between adhesion molecules (AMs) and their counter-receptors expressed on BM endothelium, migrate through endothelial cells and into the
stromal cell microenvironment. Within the BM microenvironment, the
homing process continues as HSCs, again through interactions between
AMs and cognant ligands, specifically anchor within appropriate BM
niches and begin the process of hematopoiesis. Mobilization of HSCs
from their BM niches into the periphery following administration of
growth factors or chemotherapeutic agents is likely to involve sequential loss or alterations in adhesive interactions between HSCs
and stromal cells, and then endothelium, with final release into the
periphery. Although data implicating certain AMs in various stages of
stem cell homing and egress from the BM are beginning to
accumulate,1-7 much remains to be learned regarding the
relationship between AMs and stem cell trafficking, and how this
process affects stem cell engraftment and long-term hematopoiesis in
transplanted recipients.
While AMs are likely to direct the trafficking of HSCs within the BM
microenvironment and periphery, the position of these cells in specific
phases of cell cycle is believed to dictate the hematopoietic potential
of HSCs.8 A large body of evidence in both in vitro and in
vivo systems supports the notion that mitotic quiescence is a
fundamental characteristic of HSCs and is essential for the
preservation of primitive hematopoietic function. Recent reports in the
murine,9 feline,10 and human11
systems suggest that the cell cycle of HSCs may be relatively
long and that these cells may display reduced engraftment potential at the time of active cell division.12-14 On the other hand,
few reports seem to indicate that cells capable of long-term
engraftment cycle quickly following transplantation.15
These data pose the question of whether changes in AM status,
concurrent with entry of these cells into active cell division,
influence the engraftment potential of cycling HSCs. We and others have
begun to investigate relationships between cell cycle progression and
expression or function of AMs on primitive hematopoietic progenitor
cells (HPCs). Some reports recently demonstrated an increased
expression of certain AMs16-19 and, in some cases,
increased adhesion17 of primitive HPCs in active phases of
cell cycle. These data, which clearly establish a link between cell
cycle status and AM repertoire, suggest the possible existence of
regulatory control mechanisms between expression or function of AMs and
cell cycle position of HSCs, which may in turn have an impact on the
engraftment potential of these cells.
In the present study, we examined the contribution of 6 AMs to
the homing and long-term engraftment potential of primitive HPCs using
an in vivo murine BM transplantation model. Results of these studies
imply a theoretical phenotype of a Sca-1+lin
engrafting cell as expressing high levels of CD49e and CD43, and low
levels of CD11a, CD49d, and CD62L. Cells within this AM-defined phenotype were determined to reside in G0/G1;
however, their proliferative response to in vitro cytokine stimulation
was rapid and proved to be a good predictor of long-term engraftment
potential, as did the content of high proliferative potential-colony
forming cells (HPP-CFCs) and low proliferative potential-colony forming cells (LPP-CFCs). Whether this AM phenotype imparts to
Sca-1+lin cells more efficient homing and/or
anchorage within the BM microenvironment, or enriches for HSCs within
Sca-1+lin cells, remains to be determined.
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Materials and methods |
Mice
C57BL/6 female mice (Jackson Laboratories, Bar Harbor, ME) were
purchased at 8 to 10 weeks of age and allowed to acclimate for 1 to 2 weeks prior to being used in these studies. C57BL/6 mice are
Hbbs/Hbbs (hemoglobin single), are glucose
phosphatase isoenzyme type
Gpi-1b/Gpi-1b, and
express the CD45.2 allotype. Congenic BM donors were of 2 different
strains: (1) B6 mice, which are Hbbd/Hbbd
(hemoglobin diffuse) and
Gpi-1a/Gpi-1a
(B6.Gpi-1a) and (2)
B6.SJL-PtrcaPep3b/BoyJ mice (B6.BoyJ), which
express the CD45.1 allotype. Congenic donor mice were maintained in our
breeding colony and used between 8 to 12 weeks of age. These studies
were approved by the Indiana University School of Medicine
Institutional Animal Care and Use Committee.
Flow cytometric cell sorting and analysis of
Sca-1+lin cells expressing one or
more AMs
Sca-1+lin cells were isolated from
C57BL/6 mice as previously described.20 Briefly,
low-density (1.077 g/mL or less) BM mononuclear cells were stained with
phycoerythrin (PE)-conjugated Sca-1 and fluorescein
isothiocyanate (FITC)-conjugated CD3 and CD45R/B220, and sorted by
means of a FACStarplus flow cytometer (Becton
Dickinson Immunocytometry Systems, San Jose, CA) to yield
Sca-1+lin cells. To isolate AM subfractions
of Sca-1+lin cells, mononuclear cells were
stained with Sca-1 and lineage monoclonal antibodies as above, along
with the biotinylated antibody recognizing one of the
following: CD11a (leukocyte-function-associated antigen 1 [LFA-1], clone 2D7), CD43 (Leukosialin, clone
S7), CD44 (homing-associated cell AM[HCAM], clone IM7),
CD49d (very late antigen 4 [VLA-4], clone 9C10), CD49e
(VLA-5, clone 5H10-27; MFR5), or CD62L (L-selectin, clone MEL-14). All
antibodies used in this study were obtained from Pharmingen (San Diego,
CA). AM antibodies were visualized by streptavidin-APC
(allophycocyanin) (Molecular Probes, Eugene, OR). Cells were sorted on
a FACStarplus flow cytometer as follows (Figure
1): A primary light scatter gate was
constructed and used to visualize FITC and PE fluorescence of stained
cells. A second gate was constructed to contain PE+ and
FITC cells (Sca-1+lin
cells, Figure 1A), and the APC fluorescence of these gated cells was
examined in a single-parameter histogram (Figure 1B-C). The fluorescence of a sample stained with Sca-1-PE, CD3-FITC, B220-FITC, and nonspecific biotinylated antibodies followed by streptavidin-APC was used to determine background APC fluorescence of
Sca-1+lin cells. On the basis of this
determination, sort regions were created within the APC histogram to
isolate Sca-1+lin cells that were positive or
negative for CD11a, CD43, CD49e, or CD62L (referred to as
Sca-1+lin AM+ or
Sca-1+lin AM cells hereafter)
(Figure 1B). To ensure adequate separation, cells were gated to include
the upper and lower 30% to 40% of the APC histogram, with the middle
30% to 40% of the cells discarded. In the cases of CD44 and CD49d,
where nearly 100% of Sca-1+lin cells
expressed these 2 markers (Table 1), the
brightest and dimmest 30% to 40% of
Sca-1+lin cells were sorted and referred to
as "bright" or "dim," respectively (Figure 1C). In some cases,
Sca-1+lin cells were sorted while ignoring
the signal from the AM antibody, to obtain a control group of
Sca-1+lin cells. These cells were used to
test for in vivo blocking activity of the AM antibody used for cell
sorting. Cells were maintained at 4°C throughout cell sorting and
were collected into Iscove's Modified Dulbecco's Medium (IMDM)
containing 20% fetal bovine serum (FBS) (Hyclone, Logan, UT).
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| Figure 1.
Flow cytometric cell sorting of
Sca-1+lin AM+ or AM
cells.
Low-density murine BM cells were stained with Sca-1-PE,
FITC-conjugated CD3 and B220 (lin), and biotinylated antibodies
recognizing either CD11a, CD43, CD44, CD49d, CD49e, or CD62L, followed
by the addition of streptavidin-APC. A primary light scatter gate was
constructed and used to visualize PE and FITC fluorescence (panel
A). A second gate containing PE+ and
FITC cells (Sca-1+lin cells,
panel A) was drawn, and the APC fluorescence of these gated
Sca-1+lin cells were visualized in a
single-parameter histogram (filled histograms, panels B and C).
Background APC fluorescence of Sca-1+lin
cells (open histogram, panels B and C) was determined by staining a
sample with Sca-1-PE, CD3-FITC, B220-FITC, and nonspecific
biotinylated antibodies followed by streptavidin-APC. Sort regions,
depicted by the vertical lines and arrows in panels B and C, were
created to isolate the upper and lower 30% to 40% of
Sca-1+lin cells expressing the particular AM.
For CD11a, CD43, CD49e, and CD62L, a distinct "positive" and
"negative" AM fraction could be isolated (panel B), but in the
cases of CD44 and CD49d, where nearly 100% of
Sca-1+lin cells expressed these markers, the
brightest and dimmest 30% to 40% of
Sca-1+lin cells were sorted and referred to
as "bright" and "dim," respectively. To test for in vivo
blocking activity of AM antibodies, a group of
Sca-1+lin cells were sorted from each AM
group while the APC signal from the AM antibody was ignored. Sort
regions are shown for CD49e and CD49d as representative examples. To
isolate Sca-1+lin cells on the basis of their
expression of both CD49d and CD49e,
Sca-1+lin cells were sorted to purity, panel
A. Since these cells were FITC, they were stained with
FITC-labeled CD49d and biotinylated CD49e, followed by streptavidin-APC
(panel D). Based on nonspecific background fluorescence, denoted by
dotted box in lower left of panel D, 4 sort regions were created as
follows: (1) APC FITC+ cells were gated and
sorted as CD49e CD49dbright; (2)
APC+ FITC+ cells were sorted as
CD49e+ CD49dbright; (3) APC
FITC cells were sorted as CD49e
CD49ddim; and (4) APC+ FITC cells
were sorted as CD49e+ CD49ddim (panel
D).
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To isolate Sca-1±lin cells on
the basis of their expression of both CD49d and CD49e,
Sca-1±lin cells were isolated with the use
of Sca-1-PE, CD3-FITC, and B220-FITC as described above. These cells,
which are PE+ and FITC, were then stained
with biotinylated CD49e and FITC-labeled CD49d (Figure 1D). CD49e was
developed with streptavidin-APC, and the cells were washed and
resuspended for flow cytometric cell sorting on a
FACStarplus flow cytometer. Stained
Sca-1±lin cells contained within a primary
light scatter gate were examined for their FITC and APC fluorescence in
a dual parameter dot plot, and 4 sort regions (Figure 1D), each
containing 10% to 20% of the Sca-1±lin
cells, were created as follows: (1) APC
FITC± cells were gated and sorted as CD49e
CD49dbright; (2) APC± FITC± cells
were sorted as CD49e± CD49dbright; (3)
APC FITC cells were sorted as
CD49e CD49ddim; and (4) APC±
FITC cells were sorted as CD49e±
CD49ddim. Cells were maintained at 4°C throughout cell
sorting and were collected into IMDM containing 20% FBS.
To analyze the dual expression of AM on
Sca-1+lin cells, groups of
Sca-1+lin AM+ or AM
cells were isolated as described above with the use of Sca-1-FITC, CD3-PE, B220-PE, and biotinylated-AM antibodies developed with streptavidin-APC. The resulting groups of
Sca-1+lin cells, which are negative for PE
fluorescence, were then stained with the PE-conjugate of the remaining
5 AM antibodies not used in the primary sort (ie,
Sca-1+lin CD49e+ and
Sca-1+ lin CD49e cells would be
subsequently stained with CD11a, CD43, CD44, CD49d, and CD62L). The PE
fluorescence of these stained cells was examined on a FACScan (Becton
Dickinson), and the percentage of each primary adhesion
phenotype expressing each of the other AMs was calculated on the basis
of background fluorescence of cells stained with PE-conjugated
nonspecific myeloma proteins. In every case, background PE fluorescence
of Sca-1+lin AM+ or
AM cells was less than 5% of that observed after
staining these cells with 1 of the 5 PE-conjugated AM antibodies.
Separation of cell cycle subfractions of
Sca-1+lin AM+ or
AM cells
Sca-1+linAM+ or
AM cells enriched for engraftment potential were isolated
as described above, resuspended in Hoechst buffer (Hanks' balanced
salt solution [Biowhittaker, Walkersville, MD], 20 mmol/L
HEPES [Biowhittaker], 1 g/L glucose, and 10% fetal calf serum [FCS]), and stained with Hoechst 33342 (Molecular Probes) at 10 µmol/L for 45 minutes at 37°C as previously
described.21 To limit dye efflux via the MDR-1
pump, 100µmol/L verapamil (Sigma Chemical Co, St Louis, MO) was added
to the staining buffer.22 Cells were washed and
resuspended in Hoechst buffer plus verapamil for cell sorting. Cells
falling within a primary light scatter gate and containing 2n DNA
were gated and sorted as G0/G1. Cells containing between 2n and 4n DNA were gated and sorted as
S/G2+M. Hypodiploid events, when present, were excluded
from sort windows. When quantities were sufficient, a small portion of
sorted cells were subjected to postsort analyses, either by examining
Hoechst fluorescence or by propidium iodide staining, as previously
described.23
HPP-CFC and LPP-CFC assay
Between 0.8 × 103 and 1.5 × 103
Sca-1+lin cells or AM subfractions were
suspended in triplicate in 1 mL double-layer agar cultures and assayed
for HPP-CFCs and LPP-CFCs as previously described.24 Cultures were incubated in a 100%-humidified 5% O2, 10%
CO2, and 85% N2 environment. Recombinant
hematopoietic growth factors were used as follows: 200 U/mL murine
interleukin-3 (mIL3), 1000 U/mL mIL1- , 50 ng/mL murine stem cell
factor (mSCF), 25 ng/mL murine granulocyte macrophage-colony
stimulating factor (mGM-CSF) (all from PeproTech, Rocky Hill, NJ), and
1600 U/mL human macrophage-CSF-1 (hM-CSF, Genetics Institute, Camden,
MA). On day 14, colonies larger than 0.5 mm were scored as HPP-CFC and
those smaller than 0.5mm as LPP-CFC.
Transplantation protocol
C57BL/6 female recipients between 10 and 12 weeks of age were
lethally irradiated (split dose of 700 centrigrays [cGy] followed by
350 cGy 3 to 4 hours later) from a 137Cs gamma irradiator
(GammaCell 40; Nordion International, Kanata, Ontario, Canada). Mice
were transplanted via tail vein injections 3 to 6 hours later with
0.25 × 103 to 1.0 × 103 donor
B6.Gpi-1a or B6.BoyJ cells, along with
0.3 × 105 to 1.0 × 105 competitor cells
(low-density BM cells of C57BL/6 origin), as described in Figures.
Recipient mice were bled from the tail vein monthly until 6 or 8 months
posttransplantation for analysis of donor-derived hematopoiesis.
Determination of donor chimerism in transplanted
recipients
Chimerism in C57BL/6 mice, when B6.Gpi-1a
mice were used as donors, was determined by analyzing the percentage of
donor-derived hemoglobin or Gpi-1 in peripheral blood obtained from
tail veins as previously described.25 To examine
multilineage engraftment in these mice, whole-blood samples obtained
from the retro-orbital sinus were stained separately with
FITC-conjugated CD45R/B220, or FITC-conjugated CD3, and PE-conjugated
Gr-1, lysed, and then isolated by flow cytometric cell sorting to yield
B lymphocytes, T lymphocytes, and granulocytes, respectively. These
purified lineages were then analyzed for Gpi-1
content.25
Chimerism in C57BL/6 mice receiving donor cells of B6 BoyJ
origin was determined by means of flow cytometry to calculate the percentage of CD45.2 peripheral blood cells. Whole-blood
samples obtained from tail veins were lysed and stained with
PE-conjugated CD45 and FITC-conjugated CD45.2. CD45+ cells
were gated and examined for the percentage of CD45.2
cells (cells of B6.BoyJ origin). Multilineage engraftment in these mice
was determined by staining with each of the 3 following combinations of
antibodies, CD45.2-FITC and Gr-1-PE, CD45.1-PE and CD3-FITC, and
CD45.1-PE and B220-FITC, and analyzing the percentages of donor-derived
granulocytes, T lymphocytes, and B lymphocytes, respectively.
Short-term culture
Cultures of 0.2 × 103 to 4 × 103
Sca-1+lin AM+ or AM
cells, or cell cycle subfractions of these cells, were initiated in
IMDM supplemented with 20% FBS and 2 × 105 mol/L
2-mercaptoethanol in an atmosphere of 5% CO2 in
100%-humidified air. Cytokines were delivered on day 0 as follows: 100 ng/mL mSCF (PeproTech), 500 U/mL mIL1 (Genzyme, Cambridge, MA), 100 U/mL mIL3 (Genzyme), 100 ng/mL hIL6, and 50 ng/mL human Flt3 ligand (hFlt3-L). The hIL6 and hFlt3-L were kind gifts from Amgen
(Thousand Oaks, CA) and Immunex (Seattle, WA), respectively. Care was
taken to keep the cell concentration below 1 × 106
cells/mL. Fresh cells or aliquots of cultured cells were removed on
days 1 and 2 and stained with propidium iodide for cell cycle analysis23 or incubated for an additional 11 to 14 days, at which time cells were harvested, enumerated, and analyzed by flow cytometry for the percentage of cells expressing Sca-1 with the use of
anti-Sca-1-PE.
Statistical analysis
Data are expressed as the mean ± SEM where applicable.
Differences between groups were analyzed by means of an unpaired
2-sided t test. A probability value of less than .05 was
considered significant. Regression analysis was used to analyze the
rate of exit from G0/G1 phases of the cell cycle.
| |
Results |
Expression of AMs on Sca-1+lin cells
As a first step in defining AMs important for engraftment of
primitive HPCs, we examined the pattern of expression of CD11a, CD43,
CD44, CD49d, CD49e, and CD62L on fresh
Sca-1+lin cells. As seen in Table 1,
expression of these 6 AMs was varied, with nearly 100% of
Sca-1+lin cells expressing CD44 and CD49d,
and approximately 50% expressing CD49e and CD62L. Expression of CD11a
and CD43 was observed on approximately 80% of
Sca-1+lin cells.
Primitive and mature progenitor cell content of
Sca-1+lin AM+ or
AM cells
Since HPP-CFCs likely contain some of the most primitive
HPCs,26 the HPP-CFC frequency of our 12 groups of
Sca-1+lin AM+ or AM
cells was determined. Data in Table 2
show that CD43 and CD49e were expressed on the majority of HPP-CFCs
residing in the Sca-1+lin cell fraction,
while Sca-1+lin cells lacking expression of
either CD11a or CD62L contained a higher fraction of HPP-CFCs than
Sca-1+lin CD11a+ or
Sca-1+lin CD62L+ cells (Table 2).
LPP-CFCs, progenitors more committed to lineage differentiation than
HPP-CFCs, were mostly enriched among the same AM
profile as HPP-CFCs (Table 2). To ensure that the AM antibody used for cell sorting did not induce any negative
influences on primitive HPC clonogenic activity,
Sca-1+lin cells treated with each AM antibody
but not sorted on adhesion phenotype were also assayed for HPP- and
LPP-CFC content. HPP- and LPP-CFC activity of these antibody-treated
cells was not different from that of untreated
Sca-1+lin cells (data not shown), indicating
that the AM antibodies used for cell sorting did not inhibit in vitro
activity of progenitor cells. Fractions enriched for HPP- and LPP-CFC
activity also exhibited greater cellular expansion during 11 days of in
vitro cytokine-stimulated cell culture (Table 2).
Chimerism in mice transplanted with
Sca-1+lin AM+ or
AM cells
To determine the engraftment potential of the 12 groups of
Sca-1+lin AM+ or AM
cells, a competitive repopulation assay in lethally irradiated recipient mice was performed. Chimerism in recipient mice transplanted with either total Sca-1+lin cells or
Sca-1+lin AM+ or AM
cells was evident 4 weeks posttransplantation and continued to increase
until 2 months posttransplantation, at which time chimerism stabilized
(data not shown). Figure 2A shows that
Sca-1+lin cells expressing CD43 and CD49e
appeared to be highly enriched for long-term engraftment potential, as
Sca-1+lin cells lacking expression of either
of these 2 molecules failed to provide measurable chimerism in
recipients at 6 months posttransplantation. Sca-1+lin cells expressing low levels of
CD11a, CD49d, and CD62L were superior competitors compared with their
counterparts expressing higher levels (Figure 2A). Expression of CD44
did not appear to significantly correlate with enhanced or diminished
engraftment potential of Sca-1+lin cells. As
indicated by the horizontal bars in Figure 2A, anti-AM antibodies used
for cell sorting did not interfere with engraftment of
Sca-1+lin cells, as mice transplanted with
Sca-1+lin cells treated with each
anti-AM antibody exhibited chimerism similar to that of
mice receiving untreated Sca-1+lin cells. All
groups of Sca-1+lin
AM+ or AM cells contributed equally to
lineage-specific hematopoiesis, as indicated by similar levels of
donor-derived chimerism in myeloid (Gr-1+) and lymphoid
(CD3+ or B220+) cells (data not
shown).
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| Figure 2.
Percentage of donor-derived chimerism in transplanted
mice.
Percentage of donor-derived chimerism in mice
transplanted with Sca-1+lin cells,
Sca-1+lin AM+ or AM
cells (panel A), or Sca-1+lin cells
fractionated with the use of CD49d and CD49e simultaneously (panel B).
Lethally irradiated C57BL/6 mice were transplanted with
1 × 103 total Sca-1+lin cells
(hatched bar), Sca-1+lin
AM+ (black bars), or
Sca-1+lin AM (gray
bars), all of B6.Gpi-1a origin, along with
3 × 104 C57BL/6 competitor cells. Chimerism was
monitored monthly by hemoglobin analysis of peripheral blood cells.
Horizontal black lines represent mean engraftment in mice transplanted
with "antibody control" cells (Sca-1+lin
cells treated with the AM antibody but not sorted for the AM
phenotype). Data are expressed in panel A as the mean ± SEM
of 3 to 12 mice per group from 1 to 5 separate experiments (analyzed at
6 months posttransplantation), and in panel B as the mean ± SEM
of 3 to 5 mice per group in 1 experiment. Trends in engraftment similar
to that seen in panel A were obtained in experiments in which B6.BoyJ
donor cells were used along with 1 × 105 C57BL/6
competitor cells. *The presence of statistical significance between the
AM+ and AM fraction of
Sca-1+lin cells; P < .05. **The
presence of statistical significance between
Sca-1+lin CD49e+
CD49ddim cells and each of the other 3 phenotypes at 8 months posttransplantation.
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The importance of CD49e expression in fractionating
Sca-1+lin cells into engrafting phenotypes is
illustrated in Figure 2B. With the use of both CD49e and CD49d,
Sca-1+lin cells were further fractionated to
yield 4 groups of cells (each being either positive or negative for
either marker). All 4 groups of cells were analyzed for their
engraftment potential in the same competitive repopulation assay
described above. Figure 2B shows that chimerism in mice transplanted
with Sca-1+lin cells expressing a
CD49e+ CD49ddim phenotype was higher than that
in mice transplanted with similar cells exhibiting a CD49e+
CD49dbright phenotype, while mice receiving either group of
Sca-1+lin cells devoid of CD49e expression
had undetectable engraftment. These results are consistent with data in
Figure 2A, in that Sca-1+lin cells that were
CD49e+ or CD49ddim were better competitors than
their CD49e or CD49dbright counterparts,
respectively. Mice receiving Sca-1+lin
CD49e+ CD49dbright cells exhibited transient
chimerism at 2 months posttransplantation that diminished after several
months, suggesting that this phenotype consisted of cells capable of
short-term engraftment only. Sca-1+lin cells
exposed to CD49e and CD49d antibodies provided 50% ± 8% chimerism
in recipients, a figure not statistically different from that provided
by untreated Sca-1+lin cells
(67.8% ± 11% chimerism, P > 0.1) (Figure 1A). Taken
together, these results suggest a theoretical phenotype of a
Sca-1+lin engrafting cell as being positive
for expression of CD43 and CD49e, but negative or low for expression of
CD11a, CD49d, and CD62L.
Analysis of AM expression on engrafting vs nonengrafting phenotypes
of Sca-1+lin cells
To better identify AMs potentially important in
engraftment, subfractions of Sca-1+lin cells
demonstrated as having enriched engraftment potential were surveyed for
their repertoire of AM expression in comparison with their
nonengrafting counterparts. Figure 3
shows that expression of CD11a (panel A), CD49dbright
(panel D), and CD62L (panel F) was slightly lower on engrafting than on
nonengrafting phenotypes, while expression of CD43 and CD49e was
greater on engrafting cells. Interestingly, expression of CD43 (panel
B) was more than 4 times greater on engrafting phenotypes defined by
CD11a and CD49e, and expression of CD49e (Panel E) was more than 2- and
5-fold greater on engrafting phenotypes defined by CD11a and CD43,
respectively; this illustrates once again the importance of CD43 and
CD49e in engraftment. Examination of mean channel fluorescence and peak
channel fluorescence also revealed high levels of expression of CD43
and CD49e on engrafting phenotypes of
Sca-1±lin cells (data not shown). Expression
of CD44bright (Figure 3C) was slightly increased on
engrafting phenotypes.
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| Figure 3.
Expression of AMs on engrafting and nonengrafting
phenotypes of Sca-1+lin cells.
Sca-1+lin cells were subfractionated by cell
sorting into AM+/AMbright or
AM/AMdim groups with the use of CD11a ( ),
CD43 ( ), CD49d ( ), CD49e ( ), and CD62L ( ) to give
engrafting or nonengrafting phenotypes, designated E and NE,
respectively. CD44 was omitted in the primary sort since this marker
was not useful in defining engrafting phenotypes (Figure 2). These
resulting 10 groups of cells were phenotyped for their expression of
other AMs not used in the primary sort (listed at the top of each
scatter plot), and the percentage of each engrafting (E) and
nonengrafting (NE) phenotype positive for expression of other AMs was
calculated after subtracting nonspecific background fluorescence. Each
individual data point represents the percentage of positive expression;
horizontal bars are mean expression. For example, the open circles in
Panel A represent the expression of CD11a on CD49e+ cells
(E) and CD49e cells (NE). In panel B, open squares
represent expression of CD43 on CD11a cells (E) and
CD11a+ cells (NE), also shown in panel G. Since
100% of Sca-1+lin cells express CD44 and
CD49d (Table 1), only the proportion of engrafting and nonengrafting
phenotypes positive for CD44bright and
CD49dbright expression was calculated. The histogram in
panel G illustrates the high level of expression of CD43 (x-axis) on
Sca-1[+lin CD11a cells (open
histogram) relative to CD11a+ cells (filled histogram),
while panel H shows the high level of expression of CD43 on
Sca-1+lin CD49e+ cells (open
histogram) relative to CD49e cells (filled histogram).
Data are from 1 of 2 experiments with similar results.
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Cell cycle status
Since HSCs are believed to represent relatively quiescent cells,
the cell cycle status of the 12 phenotypes of
Sca-1+lin AM+ or AM
cells was evaluated to determine whether each of the AM subfractions of
Sca-1+lin cells enriched for engraftment
potential would also be more quiescent than the corresponding
nonengrafting phenotype. However, 5 out of 6 Sca-1+lin fractions that were negative or dim
for expression of AMs were significantly more quiescent than the
AM+ fraction (Figure 4). In
the cases of CD49d and CD62L, the engrafting phenotype was more
quiescent than the nonengrafting, but in the cases of CD43 and CD49e,
the engrafting phenotype contained the higher percentage of cycling
cells (Figure 4).
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| Figure 4.
Cell cycle status of fresh
Sca-1+lin AM+ or AM
cells or total Sca-1+lin cells.
Fractions of Sca-1+lin AM+ (black
bars), Sca-1+lin AM (gray
bars), or total Sca-1+lin cells (horizontal
line) were stained with propidium iodide and analyzed on a FACScan flow
cytometer for cell cycle status as previously described.23
Data are reported as the percentage of cells in
G0/G1 phase of the cell cycle (mean ± SEM; n = 6 to 7 for every group). *The presence of statistical
significance when the AM fraction is compared with its
AM+ counterpart; P < .05.
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The cell cycle status of Sca-1+lin cells
fractionated with the use of both CD49e and CD49d expression also
correlated with the level of expression of these 2 AMs.
Sca-1+lin cells lacking expression of both
markers were more quiescent (98.6% ± 0.4% in
G0/G1) than Sca-1+lin
cells positive for the expression of both CD49e and CD49d
(78.3% ± 2.8% in G0/G1). The degree of
quiescence of Sca-1+lin cells lacking
expression of one but not both markers fell between that of
double-positive and double-negative cells
(CD49e+/CD49ddim = 93.2% ± 1.9%
G0/G1;
CD49e/CD49dbright = 96.0% ± 2.0%
G0/G1; n = 2 for all cell cycle analyses).
Cell cycle progression of AM subfractions of
Sca-1+lin cells
Rapid in vivo proliferation of HSCs following transplantation has
been reported to correlate with long-term hematopoietic function of
transplanted cells.27 We have already shown that cellular
expansion in vitro (Table 2) correlates with long-term engraftment
capability of Sca-1+lin AM+ or
AM cells (Figure 2). Differences in proliferative
potential between engrafting and nonengrafting phenotypes may be
related to the rate at which these cells exit
G0/G1 in vitro, as shown in Figure 5. AM fractions enriched for engraftment
potential, regardless of their relative degree of quiescence on day 0, exited G0/G1 more rapidly than nonengrafting
phenotypes (Figure 5). Cell cycle progression of
Sca-1+lin cells subfractionated with the use
of both CD49e and CD49d also correlated with the engraftment potential
of these cells. Sca-1+lin cells most enriched
for engraftment potential among these 4 phenotypes (CD49e+/CD49ddim) exited
G0/G1 more rapidly (slope =  25) than cells
lacking expression of CD49e (slopes = 2 to 11), or
Sca-1+lin cells expressing both CD49e and
CD49d cells (slope = 16).
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| Figure 5.
Exit from G0/G1 phases of cell
cycle.
Exit from G0/G1 phases of cell cycle of total
Sca-1+lin cells ( ), or engrafting
phenotypes (dotted lines) and nonengrafting phenotypes (solid lines) of
Sca-1+lin AM+ () and
AM () cells. Up to 4 × 103 cells/mL
were supplemented with 20% FBS, 2 × 105 mol/L
2-mercaptoethanol, 100 ng/mL mSCF and hIL6, 500 U/mL mIL1, 100 U/mL
mIL3, and 50 ng/mL hFlt3-L. Fresh cells and aliquots of cultured cells
removed on days 1 and 2 were stained with propidium iodide and analyzed
for cell cycle status as previously described.23 Data are
expressed as the mean ± SEM; n = 2 to 3 for each time point.
Slopes of linear regression lines generated from these data are
indicated next to the appropriate lines to illustrate relative rates of
exit from G0/G1 of the different populations of
cells. *The presence of statistical significance in slope of regression
line of engrafting phenotype when compared with the slope generated
from its nonengrafting counterpart; P < .05.
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Engraftment potential of Sca-1+lin
AM+ or AM cells subfractionated on the basis
of position in cell cycle
The relatively high percentage of cycling cells in engrafting
phenotypes defined as CD43+ and CD49e+ of
Sca-1+lin cells (Figure 4) led us to question
whether quiescent or cycling cells within the AM-defined engrafting
phenotypes were contributing to engraftment potential and in vitro
hematopoiesis of these cells. To this end, engrafting phenotypes of
Sca-1+lin AM+ or AM
cells were subfractionated into G0/G1 and
S/G2+M fractions with the use of Hoechst 33342 and examined
for their engraftment potential. Figure 6
shows chimerism in mice transplanted with G0/G1
or S/G2 + M fractions of total
Sca-1+lin cells,
Sca-1+lin CD11a,
Sca-1+lin CD49ddim, and
Sca-1+lin CD49e+ cells.
G0/G1 cells, regardless of which AM was used in
fractionation, provided greater levels of chimerism than equal numbers
of S/G2+M cells (P < .005), illustrating the
enhanced engraftment potential of quiescent cells.
G0/G1 cells also provided higher levels of chimerism than equal numbers of total
Sca-1+lin AM subfraction (data not shown),
negating any suggestion that the Hoechst dye may negatively influence
the function of primitive HPCs in active phases of cell cycle. Of
interest in these analyses is that, although a relatively large
percentage of Sca-1+lin CD49e+
cells were in active phases of cell cycle (Figure 4), only those in
G0/G1 accounted for the majority of the
engraftment potential of this phenotype. All groups of cells
contributed equally to lineage-specific hematopoiesis (myeloid and
lymphoid; data not shown). Table 3 shows
that the quiescent fractions of engrafting Sca-1+lin cells possessed greater
proliferative potential and gave rise to greater numbers of
Sca-1+ cells during 14 days of in vitro cytokine-stimulated
cell culture than engrafting cells in active phases of cell
cycle.
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| Figure 6.
Percentage of donor-derived chimerism of cell cycle
subfractions.
Percentage of donor-derived chimerism at 6 months posttransplantation
in mice receiving G0/G1 or S/G2+M
subfractions of total Sca-1+lin cells (),
Sca-1+lin CD11a cells (),
Sca-1+lin CD49ddim cells (),
or Sca-1+lin CD49e+ cells ().
Total Sca-1+lin cells or CD11a,
CD49ddim, or CD49e+ subfractions of
Sca-1+lin cells were isolated by cell sorting
and then subfractionated by means of Hoechst 33342 into
G0/G1 or S/G2+M fractions. Between
0.4 to 1.0 × 103 sorted donor cells from B6.BoyJ mice
were transplanted into lethally irradiated C57BL/6 mice along with
1.0 × 105 C57BL/6 competitor cells. Chimerism was
monitored monthly by CD45.2 analysis of peripheral white blood cells.
Purity of sorted donor G0/G1 cells was greater
than 97%; purity of donor S/G2+M cells was greater than
82%. Data points are values for the percentage of chimerism for
individual mice; horizontal bars are mean chimerism levels. n = 10
for G0/G1 mice, n = 12 for S/G2+M
mice. *The presence of statistical significance between mice
transplanted with G0/G1 and S/G2+M
fractions of engrafting phenotypes; P < .005.
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Table 3.
Fold-increase in total cells and Sca-1+ cells
in cultures initiated with engrafting phenotypes of
Sca-1+linAM+ or AM
cells fractionated on the basis of position in cell cycle
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Discussion |
In this report, we define a theoretical phenotype of a
Sca-1+lin long-term engrafting cell as a
quiescent cell that expresses high levels of CD43 and CD49e, expresses
low or negligible levels of CD11a, CD49d, and CD62L, and proliferates
rapidly in vitro following cytokine stimulation. These data
are a first step in defining AMs on primitive quiescent HPCs
potentially important in the homing and/or anchorage of
long-term engrafting cells within the BM microenvironment.
To the best of our knowledge, antibodies used in these studies were
nonblocking. In mice where long-term engraftment potential was
predominantly among AM/AMdim phenotypes
(CD11a, CD49d, CD62L), testing of blocking activity was crucial since
low chimerism in AM+ mice may have resulted from blockade
of homing or |
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Abstract
Introduction