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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4106-4117
Rhodamine-123 Staining in Hematopoietic Stem Cells of Young Mice
Indicates Mitochondrial Activation Rather Than Dye Efflux
By
MiJung Kim,
Donna D. Cooper,
Stanley F. Hayes, and
Gerald J. Spangrude
From the Departments of Pathology, Division of Cell Biology and
Immunology, and Medicine, Division of Hematology/Oncology, University
of Utah, Salt Lake City, UT; and Microscopy Branch, National Institute
of Allergy and Infectious Diseases, National Institutes of
Health, Rocky Mountain Laboratories, Hamilton, MT.
 |
ABSTRACT |
Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a
quiescent hematopoietic stem cell (HSC) population in mouse bone
marrow, which provides stable, long-term hematopoiesis after
transplantation. Rh-123 labels mitochondria with increasing intensity
proportional to cellular activation, however the intensity of staining
also correlates with the multidrug resistance (MDR) phenotype, as
Rh-123 is a substrate for P-glycoprotein (P-gp). To address the
mechanisms of long-term repopulating HSC discrimination by Rh-123,
mouse bone marrow stem and progenitor cells were isolated based on
surface antigen expression and subsequently separated into subsets
using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function. We determined the cell
cycle status of the separated populations and tested for HSC function using transplantation assays. Based on blocking studies using MDR
modulators, we observed little efflux of Rh-123 from HSC obtained from
young (3- to 4-week-old) mice, but significant efflux from HSC derived
from older animals. A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or
membrane potential indicated that mitochondrial activation is more
important than either mitochondrial mass or MDR function in defining
HSC in young mice. This conclusion was supported by morphologic studies
of cell subsets separated by Rh-123 staining.
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INTRODUCTION |
HEMATOPOIESIS PROCEEDS throughout life.
To maintain normal numbers of blood cells, millions of mature cells are
generated daily. This rate of production requires a progenitor
compartment that proliferates extensively. Hematopoietic stem cells
(HSC) produce progenitors for all hematopoietic lineages and are the source of the cellular elements of the blood over the life span of the
organism.
In certain strains of mice, the engrafting HSCs can be entirely
localized within a population of cells characterized by low-level expression of the T-lymphocyte antigen Thy-1.1, low or no detectable expression of a panel of markers specific for differentiated
hematopoietic lineages, and high-level expression of the Ly-6A/E
(Sca-1) antigen.1 Staining these cells with the vital
mitochondrial dye rhodamine-123 (Rh-123) can resolve two functionally
distinct subsets.2 The Rh-123high population
only transiently repopulates hematopoietic cell lineages, while the
Rh-123low stem cell population permanently reconstitutes
hematopoiesis.3-5
Two mechanisms have been proposed for the ability of Rh-123 to
discriminate functional subsets of HSC. Because Rh-123 stains mitochondria with increasing intensity as cells become
activated,6 the probe may detect a reduced mitochondrial
activation state in quiescent long-term repopulating
cells.7 Decreased intracellular accumulation of Rh-123 also
results from efflux of the dye, mediated by multidrug resistance (MDR)
genes such as P-glycoprotein (P-gp).8 Rh-123 is a known
substrate for P-gp and has been used extensively as an indicator for
P-gp activity.
P-gp is a family of plasma membrane glycoproteins encoded by three
genes in the mouse. Two genes, mdr1a and mdr1b, encode proteins that
function as energy-dependent efflux pumps9; the mdr2 gene
product may function as a phospholipid transporter important in normal
hepatobiliary function.10 The mouse mdr1a and mdr1b
isoforms and their human homologue MDR1 transport structurally diverse
molecules out of cells and have similar substrate specificities and
sensitivities to pharmacologic modulators.11
A second multidrug transporter, the multidrug-resistance associated
protein (MRP), has recently been characterized.12 MRP differs from P-gp in substrate specificity and susceptibility to
pharmacologic modulators. Rh-123 has been shown to be a relatively specific substrate for P-gp, but not MRP,13 even though
expression of MRP confers resistance to Rh-123 toxicity.14
Verapamil is an efficient inhibitor of MDR-mediated drug efflux. A
green fluorescent derivative of this drug, Bodipy-verapamil (BodVer)
has been shown to function as an MDR substrate without significant MDR
inhibition.15 BodVer preferentially accumulates in the
lysosomes of normal, drug-sensitive NIH 3T3 cells, but is rapidly
transported out of MDR cells. Therefore, separation of HSC based on
accumulation of BodVer should reflect only MDR function and lysosomal
content without being affected by mitochondrial characteristics.
Other fluorescent probes are potential candidates for assessing the
roles of MDR function versus mitochondrial membrane potentials in
defining repopulating HSC. Nonyl acridine orange (NAO), a probe that
stains mitochondria independently of their energetic state, is an
indicator of mitochondrial mass.16 Because NAO has also been shown to be an MDR substrate,17 separations of HSC
using this probe would reflect mitochondrial mass and MDR function, but
not mitochondrial activation. JC-1 is a cationic, dual emission, membrane potential-sensitive mitochondrial probe.18 The
green fluorescent monomer forms red fluorescent "J-aggregates" at
high concentrations. At appropriate dye concentrations, the emission wavelength of JC-1 fluorescence in mitochondria is an indicator of
mitochondrial membrane potential.19 It has not been
previously reported that JC-1 is an MDR substrate.
To address the relationship between MDR function, mitochondrial
membrane potential, and functional heterogeneity in the HSC compartment, we isolated stem and progenitor cells from normal mouse
bone marrow and separated subsets of these cells using fluorescent probes, which are sensitive to MDR function alone (BodVer) or mitochondrial characteristics and MDR function (Rh-123, JC-1, and NAO).
To further address the role of MDR activity in defining repopulating
HSC, we performed separations in the presence and absence of MDR
blockers. We measured the cell cycle status of each separated
cell population and tested for HSC function in transplantation assays.
The results suggest that in young mice, mitochondrial characteristics
play a greater role in defining primitive HSC than does MDR function.
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MATERIALS AND METHODS |
Animals.
C57BL-Thy-1.1/Ly-5.1 double congenic mice, C57BL/6J (B/6;
Thy-1.2/Ly-5.2/Ly-1.2), and C57BL/6-Alpha-17 (Alpha;
Thy-1.2/Ly-5.2/Ly-1.1) mice were bred and maintained in the University
of Utah Animal Resource Center. All animals were maintained on
acidified (pH 2.5) drinking water and autoclaved chow (Purina Mills
Inc, St Louis, MO) ad libitum.
Cell preparation.
Cells were prepared using Hanks' Balanced Salt Solution (HBSS)
containing 5% fetal calf serum. Bone marrow cells were prepared from
young C57BL-Thy-1.1/Ly-5.1 mice (3 to 4 weeks old except as indicated)
by crushing femora and tibia in HBSS using a mortar and pestle. Lymph
node cells were prepared from pooled inguinal, axillary, brachial, and
cervical lymph nodes by gentle teasing with forceps. Both samples were
subjected to repeated pipetting and filtered through nylon mesh (85 µm, Small Parts Inc, Miami Lakes, FL) to remove connective tissue and
debris.
Fluorescent dye uptake assay.
A leukemic cell line (K562) overexpressing the human P-gp isoform MDR1
was kindly provided by Dr Igor Roninson, University of Illinois,
Chicago, IL. The parental K562 cells, which have no detectable P-gp
mRNA,20 were obtained from the American Type Culture
Collection (Rockville, MD). Cells were maintained in phenol red-free
RPMI 1640 medium containing 10% fetal calf serum. To evaluate whether
probes were MDR1 substrates, 0.5 × 106 cells/mL K562
or K562-MDR cells were stained in the presence and absence of the MDR
blockers verapamil (50 µg/mL) or cyclosporin A (20 µg/mL). All
fluorescent probes were obtained from Molecular Probes, Eugene, OR and
used at the following concentrations: Bodipy-verapamil (BodVer), 100 nmol/L; JC-1, 5 µmol/L; NAO, 10 nmol/L; Rh-123, 250 nmol/L. Cells
were maintained at 37°C in the presence of the individual probes,
and 1-mL samples were withdrawn every 20 minutes for 3 hours. Samples
were mixed with 2 mL cold phosphate-buffered saline (PBS) containing 50 µg/mL verapamil to stop efflux of probe and were centrifuged at
0°C for 5 minutes at 1,200 rpm. Cells were resuspended in 1 mL cold
PBS containing 50 µg/mL verapamil before analysis for mean
fluorescence intensity using a FACScan instrument (Becton Dickinson
Immunocytometry Systems, San Jose, CA).
Hematopoietic stem and progenitor cell enrichments.
Bone marrow cells were magnetically lineage-depleted and subsequently
stained with fluorescein-conjugated anti-Thy-1.1,
phycoerythrin-conjugated anti-Sca-1, and biotin-conjugated
anti-Sca-2, followed after a wash with streptavidin-Red 613 (GIBCO-BRL; Life Technologies, Inc, Grand Island, NY) as previously
described.21 A FACS-Vantage instrument (Becton Dickinson
Immunocytometry Systems) was used for sorting Thy-1.1low
Sca-1+Sca-2 stem and
progenitor cells from the lineage-depleted bone marrow population.
Sorted cells were collected by centrifugation, and a small sample (10%
of the total yield) was removed for evaluation of sort purity (usually
>90%).
Staining and efflux of Rh-123, BodVer, NAO, and JC-1.
The sorted stem and progenitor cells were resuspended in 37°C HBSS
containing 200 nmol/L Rh-123, 500 nmol/L BodVer, 2 nmol/L NAO, or 400 nmol/L JC-1. After a 20-minute incubation at 37°C, the cells were
collected by centrifugation, resuspended, and incubated at 37°C for
20 minutes in HBSS. The cells were then centrifuged, resuspended in 0.5 mL HBSS, and held at 4°C during the second fluorescence-activated
cell sorting (FACS). When included in the experiment, MDR
modulators were added to each phase of the staining and efflux at
concentrations predetermined to block Rh-123 uptake by Chinese hamster
ovary cell lines overexpressing mouse mdr1a or mdr1b isoforms
(verapamil, 25 µg/mL; reserpine, 10 µg/mL; cyclosporin A, 10 µg/mL; cell lines were generously provided by Dr Philippe Gros,
McGill University, Montreal, Quebec,
Canada).11 The modulators were also confirmed
to be nontoxic to bone marrow cells in methylcellulose colony assays
when used at the above concentrations.
Second FACS sort.
The sorted stem and progenitor cells were sorted a second time,
selecting for the dullest and brightest cells in the Rh-123, BodVer,
NAO, or JC-1 staining distribution. Reanalysis of the sorted cells
showed a high level of purity (>97%).
Rh-123low/Rh-123high,
BodVerlow/BodVerhigh,
NAOlow/NAOhigh, and
JC-1low/JC-1high cells were sorted as
populations into glass tubes, and dilutions were made based on the
electronic count of the cell sorter to obtain the desired number of
cells. In some experiments, an automatic cell deposition unit (Becton
Dickinson Immunocytometry Systems) was used to deposit the required
number of cells into the wells of a 96-well microtiter plate, with each
well also containing 105 normal B6-Thy-1.2/Ly-5.2 bone
marrow cells in 200 µL HBSS. The contents of the wells were then
collected in 1-cc insulin syringes (Becton Dickinson, no. 9410) and
quantitatively transferred into recipient mice.
Irradiations and reconstitutions.
Bone marrow recipient animals (B/6 or Alpha) were exposed to 13 Gy of
radiation from a 137Cs source (Mark I gamma irradiator;
J.L. Sheperd and Associates, Glendale, CA) at a dose rate of 0.5 Gy/min. The dose was delivered in two equal fractions separated by a
3-hour rest as previously described.22
Analysis of transplant recipients.
At various times after transplantation, peripheral blood samples were
collected for immunofluorescent staining. Donor- and host-derived cells
were distinguished with monoclonal antibody (MoAb) specific for the two
alleles of Ly-5.23 Dual-color immunofluorescence, using
fluorescein-conjugated anti-Ly-5 reagents specific for the donor
alleles and biotin conjugates of antibodies specific for T-cell,
B-cell, and myeloid lineages, was used to phenotype donor-derived cells
as previously described.24
Marrow repopulating assay (MRA).
Approximately 2,000 cells of each of the Rh-123, JC-1, or NAO-separated
populations were transplanted into lethally irradiated B/6 or Alpha
mice. Bone marrow from these animals or untransplanted controls was
harvested 12 to 13 days later, and cellular reconstitution was
evaluated by determining the number of cells per femur. These cells
were then transplanted into a second set of irradiated recipients for
determination of the frequency of day 13 spleen colony-forming units
(CFU-S) per femur of each recipient group as previously described.25
Cell cycle analysis.
Sorted cell suspensions were fixed in methanol and stained in a
PBS-EDTA solution containing 1% Triton X-100, 100 U/mL RNAse, and 5 mg/mL propidium iodide before analysis for DNA content by FACScan.
Cell imaging.
For transmission electron microscopy, Rh-123low and
Rh-123high cell subsets were sorted into 1.5-mL microfuge
tubes containing 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 mol/L
sodium phosphate buffer (pH 7.2) containing 0.1 mol/L sucrose. After a
1-hour fixation, cells were stained with OsO4 and uranyl
acetate, embedded in Spurr's resin, and sectioned. Silver-gold
sections were collected on naked 300 mesh copper grids and poststained
with uranyl acetate and lead heavy metal stains. Examination and
photography used a Hitachi HU11-E-1 electron microscope
(Hitachi, Ltd, Tokyo, Japan) at 75 kV using Kodak SO-163 electron image
film (Eastman Kodak, Rochester, NY). Random images of
Rh-123low and Rh-123high cells were evaluated
visually for mitochondrial distribution and activation, based on
mitochondrial staining intensity and morphology. The photomicrographs
shown (see Fig 8) are representative of 17 images of
Rh-123low cells and 32 images of Rh-123high
cells.
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| Fig 8.
Ultrastructural analysis of isolated
Rh-123low (A) and Rh-123high (B) HSC. The
original magnification was 8,200X in each case. Pronounced mitochondrial clustering is apparent in the nuclear cleft region of the
Rh-123high cell (B), correlating with the bipolar
perinuclear localization of Rh-123 staining seen in Fig 7B. Activated
mitochondria in (B) can be identified by increased electron density due
to thickening of cristae and by the characteristic structural collapse
and "wagon wheel" appearance of actively respiring mitochondria;
these features were rarely noted in micrographs of
Rh-123low cells (A). The micrographs are representative of
17 images of Rh-123low cells and 32 images of
Rh-123high cells.
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For confocal microscopic analysis, isolated viable
Rh-123low and Rh-123high cells were wet-mounted
in 5 µL of HBSS and visualized using a Bio-Rad MRC 1000 confocal
laser scanning system (Bio-Rad Laboratories, Hercules, CA) coupled to a
Zeiss Axiovert 135 microscope (Carl Zeiss, Inc, Thornwood,
NY). Excitation used an argon laser tuned to 488 nm and fluorescence
emissions were collected through a 525-nm bandpass filter. Fluorescent
and visible images were collected simultaneously through a 63X
objective and later analyzed using NIH Image software
(National Institutes of Health, Bethesda, MD).
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RESULTS |
Fluorescent labeling of
Thy-1.1lowSca-1+Lin HSC with
various probes.
To address the mechanism that allows Rh-123low staining to
identify the subset of multipotent HSC providing long-term engraftment after transplantation,3 we tested the ability of other
fluorescent probes to segregate functionally distinct HSC subsets.
Probes were selected for their ability to function as indicators of
mitochondrial characteristics and/or MDR function. Each probe
produced a spectrum of fluorescence intensity when used to label
Thy-1.1lowSca-1+Lin stem and
progenitor cells obtained from 3- to 4-week-old mice, with the range of
dullest to brightest staining (gating on the upper and lower 5% of
total cells) being 40-fold (JC-1), 30-fold (Rh-123), 20-fold (NAO), and
7-fold (BodVer) (Fig 1). Only JC-1 labeled
HSC at a duller intensity than unseparated bone marrow when equivalent
concentrations of dye were used; this indicates the absence of
preferential efflux of Rh-123, BodVer, and NAO from HSC relative to
unseparated bone marrow. The staining intensity of JC-1 in HSC reverted
to the levels seen in unseparated bone marrow samples when the staining
was performed in the presence of the MDR blockers, suggesting
preferential efflux of JC-1 by an MDR mechanism in HSC. Staining of HSC
with BodVer and NAO resulted in relatively unimodal staining
distributions with intermediate levels of fluorescence compared with
unseparated bone marrow. Rh-123 stained HSC more brightly than bone
marrow and included a discernable shoulder at the high end of the
staining distribution (Fig 1).
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| Fig 1.
Fluorescence staining intensity observed after reacting
the indicated fluorescent probes with normal bone marrow cells (shaded histograms) or
Thy-1.1lowSca-1+Lin HSC sorted
from 3 to 4 week-old donor animals (open histograms). Staining and
efflux were performed as described in Materials and Methods.
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Effect of donor age on Rh-123 efflux.
To address discrepancies of Rh-123 staining patterns in stem and
progenitor populations reported by different
laboratories,3,26 we compared bone marrow and
Thy-1.1lowSca-1+Lin HSC
staining profiles from mice of various ages. A striking effect of donor
age on efflux of Rh-123 was apparent, as shown in
Fig 2A. In all cases, the distinct
Rh-123low population observed in
Thy-1.1lowSca-1+Lin HSC
obtained from older animals could be reversed by MDR modulators (Fig
2B, and additional data not shown). MDR modulators consistently increased the fluorescence intensity of Rh-123low cells,
but not Rh-123high cells, suggesting that the
Rh-123low subset is defined by efflux of the Rh-123 probe
as previously reported.8 However, MDR modulators had little
effect on the Rh-123 staining profile of
Thy-1.1lowSca-1+Lin HSC
obtained from 3- to 4-week-old mice, or on unseparated bone marrow
cells from any age mouse (Fig 2B). These results show a specific and
pronounced effect of mouse age on Rh-123 efflux from Thy-1.1lowSca-1+Lin HSC,
similar to previous reports of upregulated P-gp function in
mouse27 and human28 T lymphocytes derived from
aged donors.
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| Fig 2.
Effect of bone marrow donor age on efflux of Rh-123.
Thy-1.1lowSca-1+Lin HSC and
normal unfractionated bone marrow isolated from groups of mice of the
indicated ages were stained with Rh-123 as described in Materials and
Methods. (A) Shaded profiles indicate Rh-123 fluorescence in normal
bone marrow cells, while solid profiles show fluorescence of
Thy-1.1lowSca-1+Lin HSC
isolated from the same bone marrow preparation. (B) Solid lines
indicate the Rh-123 fluorescence intensity of
Thy-1.1lowSca-1+Lin HSC
isolated from 10-month or 3-week-old mice, except in the indicated
panel where the solid line indicates total bone marrow cells. Stippled
profiles indicate the same cells stained with Rh-123 in the presence of
20 µg/mL cyclosporin A; similar results were obtained using verapamil
as an MDR modulator (data not shown).
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Evaluation of MDR-mediated efflux of fluorescent probes.
To test and compare the ability of probes to act as MDR substrates, we
measured the dye loading kinetics in a pair of cell lines, the human
chronic myelogenous leukemia line K562 and a sister clone
overexpressing human MDR1. To do this, we suspended the cell lines in
equal concentrations of dye and measured the increase in fluorescence
intensity with time. The activity of MDR1 in the transduced cell line
could be visualized as a decreased rate of loading relative to the
parental line, particularly when Rh-123 or JC-1 were used as substrates
(Fig 3A and B). Rh-123 and JC-1 efflux
could be partially reversed with verapamil or cyclosporin A, but even
in the presence of the blockers, there was a differential between the
parental and transfectant cell lines, suggesting that inhibition of
efflux was incomplete. An efflux mechanism endogenous to K562 cells was
detected when NAO and BodVer were used as substrates, as dye loading
into both parental and MDR1 transfectants was enhanced in the presence
of verapamil or cyclosporin A (Fig 3C and D). This effect was also
noted late in the time course of JC-1 loading (Fig 3B). K562 cells are
known to express a newly described MDR protein, the multidrug
resistance-associated protein (MRP).29 The substrate
specificity of MRP (low specificity for Rh-123 as a
substrate,13 and sensitivity to modulation by verapamil and
cyclosporin A30) fits with the interpretation that MRP is
the endogenous pump responsible for efflux of JC-1, BodVer, and NAO
seen in Fig 3B through D. Despite this endogenous efflux activity, the
differential loading of the two cell lines in the absence of modulators
confirms that both NAO and BodVer are P-gp substrates15,16
and establishes that JC-1 is also an MDR substrate. In addition, these
experiments confirm multiple efflux mechanisms to which the four probes
are differentially sensitive.13
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| Fig 3.
Determination of P-gp-mediated efflux of four
fluorescent probes. Each probe was incubated with the parental K562 or
MDR1-transfected cell line at 37°C, and mean fluorescence intensity
was measured as a function of time. Verapamil (50 µg/mL) was added to
each sample at the time of washing to prevent efflux before
fluorescence measurement. When included as a modulator of efflux,
either cyclosporin A (20 µg/mL) or verapamil (50 µg/mL) was present
during the incubation period, as well as during analysis. Symbols in
each panel represent parental K562 cells ( ,  ) or MDR1 transfected
K-562 cells ( ,  ); open symbols represent uptake in the absence of
modulators, while closed symbols indicate that either cyclosporin A (B
and C) or verapamil (A and D) was present during probe loading.
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Functional analysis of HSC separated by fluorescent mitochondrial
probes.
Thy-1.1lowSca-1+Lin HSC were
isolated from 3- to 4-week-old mice and stained with mitochondrial dyes
JC-1, NAO, or Rh-123. JC-1 and Rh-123 preferentially stain activated
mitochondria,31 while NAO stains mitochondria independently
of their energetic state.16 The brightest and dullest 30%
of cells in each staining distribution (Fig 1) were selected by cell
sorting, and 2,000 cells were transplanted into lethally irradiated
recipient mice. Twelve to 13 days later, bone marrow was harvested from
the primary recipients and transplanted into secondary recipients for
determination of MRA based on the content of CFU-S-13 in marrow
isolated from the primary recipients. As shown in
Table 1, both Rh-123 and JC-1 segregated
MRA activity into the dullest staining population. In contrast, NAO did
not segregate MRA activity between dull- and bright-staining cells. Table 1 illustrates the use of MRA for detecting primitive stem and
progenitor cells in a relatively short-term assay25
(compare the low/high ratios for marrow cellularity with those for
CFU-S/femur). Because all three probes are substrates for MDR pumps
(Fig 3), these results support the hypothesis that the ability of
Rh-123 and JC-1 to separate two classes of HSC is dependent on their ability to discriminate mitochondrial membrane potentials rather than
MDR-mediated efflux.
Selection of HSC subsets using an MDR probe.
To further address the relative roles played by MDR function and
mitochondrial activation in defining primitive HSC, we used BodVer to
isolate subsets of HSC based on MDR function in the absence of a
mitochondrial specificity. In parallel, we separated the same cell
preparations using Rh-123. To establish relative repopulating abilities
of HSC stained at low and high levels with each probe, we performed
competitive repopulation assays with each population. As shown in
Fig 4, long-term repopulating activity was
concentrated in the Rh-123low population, as 100% of
animals transplanted with 50 of these cells were strongly reconstituted
(>30% of circulating blood cells) with donor-derived peripheral
blood cells of T, B, and myeloid lineages 12 weeks posttransplant. No
recipients of 50 Rh-123high cells were reconstituted with
donor-derived cells (not shown), while only one of eight recipients of
200 Rh-123high cells was reconstituted in multiple
hematopoietic lineages with donor-derived cells (Fig 4A).
Transplantation of 50 BodVerlow cell cells resulted in 20%
long-term repopulating efficiency (two of 10 mice were strongly
reconstituted in T, B, and myeloid lineages), while the
BodVerhigh cell population showed little or no long-term
repopulating activity after transplantation of 200 cells (Fig 4B).
Therefore, although some segregation of repopulating activity was
observed between BodVerlow and BodVerhigh
populations, selection of HSC based on efflux of BodVer did not concentrate HSC function in the low-staining fraction to the same extent as did selection with Rh-123.
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| Fig 4.
Comparison of long-term repopulating activity in
Thy-1.1lowSca-1+Lin HSC
separated using Rh-123 (A) or BodVer (B). The brightest and dullest
15% of cells in each staining distribution were isolated from Ly-5.1
bone marrow, and the indicated numbers of each population were
transplanted into lethally irradiated Ly-5.2 recipient animals in the
presence of 105 normal Ly-5.2 bone marrow cells. Each
animal (represented by each set of bars in the graphs) was bled 4, 6, 8, and 12 weeks posttransplant (time points are represented by
individual bars within each set of four bars in the graphs), and Ly-5.1
peripheral blood cells were identified and phenotyped by flow
cytometry.
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Inhibition of MDR function in HSC using MDR blockers.
We used three functionally distinct MDR blockers to address the role of
active dye efflux in determining the functional distribution of
Thy-1.1lowSca-1+Lin HSC
after staining with the fluorescent probes. Little effect was observed
when normal bone marrow cells were incubated with the individual probes
alone compared with incubations in the presence of MDR blockers
(Fig 5A through C). A very slight increase
in fluorescent intensity was observed when JC-1 staining was performed in the presence of blockers, similar to that observed with Rh-123 (Fig
2). A more profound shift was noted in BodVer or NAO intensity in the
presence of blockers (Fig 5A and B). This observation is consistent
with the data shown in Fig 3, which indicates that BodVer and NAO are
significant substrates for a non-P-gp MDR mechanism compared with
Rh-123 and JC-1. When parallel experiments were performed to evaluate
the sensitivity of purified
Thy-1.1lowSca-1+Lin HSC to
modulation of staining intensity by MDR blockers, we observed that
incubation of the probes in the presence of MDR blockers always
resulted in a shift of the staining intensity of the entire population
of Thy-1.1lowSca-1+Lin HSC
(Fig 5D through F), in marked contrast to the results obtained with
Rh-123 (Fig 2). These findings suggest that a non-P-gp efflux mechanism is selectively expressed in
Thy-1.1lowSca-1+Lin HSC
compared with the total bone marrow population. BodVer, NAO, and JC-1
are substrates for both efflux mechanisms, while Rh-123 is relatively
selective for P-gp.13 Alternatively, the modulators may
selectively alter the subcellular distribution of the probes in
Thy-1.1lowSca-1+Lin
HSC.32 However, the results shown in Fig 5 are similar to
the data shown in Fig 3, where MDR blockers enhanced uptake of BodVer into both parental and MDR1-transfected cell lines. This result is
consistent with the interpretation that a membrane pump distinct from
P-gp is selectively active in bone marrow stem and progenitor cells.
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| Fig 5.
Retention of fluorescent probes by normal bone marrow
cells (A through C) and
Thy-1.1lowSca-1+Lin HSC (D
through F) in the presence or absence of pharmacologic MDR modulators.
All plots were derived from a single experiment using 7-week-old donor
animals. Shaded histograms indicate staining with probe alone, while
open histograms indicate staining in the presence of cyclosporin A as
an MDR modulator to block dye efflux. In all cases, cells were
incubated with the probe for 20 minutes at 37°C, washed, and
incubated in the absence of probe for an additional 20 minutes at
37°C to allow efflux. Cyclosporin A, when used, was present during
both stages of the incubation; similar results were obtained using
verapamil or reserpine as MDR modulators. Cells subsets were isolated
for subsequent analysis (Table 2, Fig 6) by gating on and sorting the
dullest and brightest 15% of cells in each profile.
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Effect of MDR inhibition on the cell cycle status of HSC subsets.
To address the effects of MDR blockers on the selection of HSC subsets
using Rh-123 and BodVer, we analyzed the cell cycle status of dull and
bright cell populations isolated with both dyes. Cell populations
selected as either Rh-123low or BodVerlow were
comprised predominantly of quiescent cells ( 3% S+G2/M) while the Rh-123high and BodVerhigh populations
contained high numbers of cycling cells ( 30% in S+G2/M; Table 2). Blockade of
efflux by verapamil had little effect on the cell cycle distribution
observed in these subsets (Table 2), despite the major difference in
fluorescence intensity observed when verapamil was used to block BodVer
staining (Fig 5D). The frequency of S+G2/M cells in the
bright-staining fractions was somewhat variable between experi-ments,
but within individual experiments the values were always very similar
in the presence or absence of verapamil. In contrast, cyclosporin A and
reserpine had little effect on the frequency of cycling cells in the
dull-staining populations, but had a pronounced effect on this
parameter in bright-staining cells (Table 2). Because of this effect
and other potential effects of cyclosporin A,32 we used
verapamil to inhibit MDR function in subsequent cell enrichments.
Verapamil blockade does not markedly change long-term engraftment by
HSC selected using either Rh-123 or BodVer.
To further investigate the role of MDR function in the selection of HSC
subsets using Rh-123 and BodVer, we performed another competitive
repopulation experiment. Rh-123low cells, isolated in the
presence of verapamil, mediated long-term repopulation in eight of 10 animals, and all positive animals were reconstituted in T, B, and
myeloid lineages at a level equal to or greater than 40% of the total
peripheral blood cells (Fig 6A). This
result is comparable to results from cell selections performed in the
absence of verapamil, where 10 of 10 animals were reconstituted (Fig
4A). Addition of verapamil to the selection protocol did not influence
the failure of Rhhigh cells to mediate reconstitution. The
recovery of long-term repopulating cells after selections using BodVer
was also not influenced by verapamil, as two of 10 animals transplanted
with 50 BodVerlow cells engrafted regardless of the
presence of verapamil (Fig 6B, compare with Fig 4B).
BodVerhigh cells isolated in the presence of verapamil
provided only marginal reconstituting activity at a dose of 50 cells
(Fig 6B).
View larger version (50K):
[in this window]
[in a new window]
| Fig 6.
Comparison of long-term repopulating activity in
Thy-1.1lowSca-1+Lin stem cells
separated using Rh-123 (A) or BodVer (B) in the presence of verapamil
as an MDR inhibitor. The brightest and dullest 15% of cells in each
staining distribution was isolated from Ly-5.1 bone marrow and
transplanted into lethally irradiated Ly-5.2 recipient animals in the
presence of 105 normal Ly-5.2 bone marrow cells. Each
animal (represented by each set of bars in the graphs) was bled 4, 6, 8, and 12 weeks posttransplant (time points are represented by each
individual bar in the graphs), and Ly-5.1 peripheral blood cells were
identified and phenotyped by flow cytometry.
|
|
Morphologic analysis of HSC subsets isolated by differential Rh-123
staining.
We used laser-scanning confocal analysis and electron microscopy to
characterize the morphology of cell populations recovered after
selection by Rh-123 staining of
Thy-1.1lowSca-1+Lin HSC.
Confocal microscopy showed the intracellular distribution of Rh-123 to
be rather uniform within Rh-123low cell populations
(Fig 7A). Of 38 Rh-123low cells
observed, 33 exhibited little or no evidence of localized accumulation
of the dye as shown in Fig 7A, while five of 38 cells showed evidence
of bipolar, perinuclear dye accumulation. In contrast, the
Rh-123high cells consistently exhibited accumulation of
Rh-123 in a bipolar fashion, within the nuclear cleft and on the
opposite side of the nucleus (Fig 7B). Optical sections at 3-µm
intervals showed the bipolar staining pattern in 28 of 28 Rh-123high cells, with the maximal staining intensity often
occurring at different section intervals for the two sites of dye
accumulation (Fig 7B and data not shown). Ultrastructural analysis of
the two subsets of HSC (Fig 8) showed
pronounced clustering of activated mitochondria of
Rh-123high cells in 27 of 32 sections evaluated, while
little activation was evident in sections of Rh-123low
cells (clustered or activated mitochondria evident in four of 17 sections evaluated). The mitochondrial distribution evident in the
ultrastructural analysis shown by representative micrographs in Fig 8
is consistent with previously published micrographs of HSC derived from
bone marrow33 and peripheral blood34 and is
also consistent with the interpretation that the distribution of Rh-123
fluorescence observed in the confocal images of these cells (Fig 7)
corresponds to mitochondrial accumulation of the dye.
View larger version (97K):
[in this window]
[in a new window]
| Fig 7.
Laser-scanning confocal microscopy of isolated
Rh-123low (A) and Rh-123high (B) HSC. In each
pair of images, the top frame was captured from transmitted light,
while the bottom frame was a simultaneous fluorescent image. Identical
magnifications (63X oil objective), illumination, and signal processing
conditions were used to image the two cell types, assuring that the
images are representative of the differential size and fluorescence
intensity of the cells. Fluorescence images were inverted and
pseudocolored so that increasing fluorescence intensity is indicated
from blue to red. Note the relatively homogeneous cytoplasmic staining
in the Rh-123low cells compared with the intense bipolar
perinuclear staining seen in Rh-123high cells. The images
are representative of 38 Rh-123low cells and 28 Rh-123high cells evaluated in two separate experiments.
|
|
| |
DISCUSSION |
Rh-123, a cationic fluorescent dye, accumulates selectively in the
mitochondria of eukaryotic cells.6 The localization of
Rh-123 to mitochondria is thought to depend on the negatively charged
membrane potential across the inner mitochondrial membrane. Rh-123 is
also a substrate for P-gp, and recent studies have suggested a
prominent P-gp activity in primitive HSC based on dye-efflux experiments.8,35,36 We designed experiments to address the relative importance of these two mechanisms in defining HSC.
We observed that efflux of Rh-123 from
Thy-1.1lowSca-1+Lin HSC
increases as a function of age (Fig 2). Very little efflux was noted
from cells obtained from 3- to 4-week-old mice, indicating that P-gp
activity is minimal in young mice. Nevertheless, Rh-123 separation of
cell subsets from these animals, in the absence (Fig 4) or the presence
(Fig 6) of MDR blockers, resulted in a profound enrichment of long-term
repopulating cells in the Rh-123low population. To further
characterize the respective roles of mitochondrial retention versus
P-gp-mediated efflux of Rh-123 in identifying primitive HSC, we used a
panel of fluorescent probes to isolate subsets of
Thy-1.1lowSca-1+Lin cells
for subsequent analysis of HSC activity. Ideally, one would use probes
selective for either mitochondrial membrane potential or MDR-mediated
efflux. However, every mitochondrial probe we have evaluated to date is
also a substrate for MDR efflux (Fig 3 and additional data not shown).
Of those tested in this study, only the probes selective for
mitochondrial membrane potential (Rh-123 and JC-1) effectively
separated and enriched primitive HSC from the
Thy-1.1lowSca-1+Lin cells.
Selections based on retention of the MDR substrate BodVer did not
enrich long-term repopulating HSC to the same extent as those based on
Rh-123 (Fig 4). While JC-1 exhibited efflux properties similar to those
of BodVer and NAO when evaluated in HSC (Fig 5D through F), only JC-1
was able to mimic the ability of Rh-123 to enrich primitive HSC (Table
1).
Very little change in the fluorescence intensity of HSC was observed
when Thy-1.1lowSca-1+Lin
cells from 3- to 4-week-old mice were stained with Rh-123 in the
presence of verapamil or cyclosporin A (Fig 2), in contrast to the
major shift in the retention of BodVer (Fig 5). This observation suggests the expression of a non-P-gp efflux pump in mouse HSC. Interestingly, while the blockers reversed MDR-mediated efflux of
BodVer, there was no effect on the frequency of long-term repopulating HSC when Thy-1.1lowSca-1+Lin
cells were fractionated under these conditions (Fig 6). This result may
be due to an incomplete blockade of efflux, or to selective accumulation of BodVer in the lysosomal compartment15
rather than specific efflux of the probe. Verapamil also failed to
block the ability of Rh-123 to localize long-term repopulating HSC in the dull-staining population. It is possible that the verapamil blockade was incomplete, allowing residual P-gp-mediated efflux of the
probe. However, because P-gp activity in cells from young mice is
minimal and because the Rh-123low population was completely
shifted into the major staining peak in the presence of verapamil (Fig
2), the evidence supporting this interpretation is not compelling.
Overall, the most conservative interpretation of our data is that in
young mice, P-gp activity is minimal in HSC and functional segregation
of long-term repopulating cells by Rh-123 is due to selective staining
of activated mitochondria.
A number of studies have shown that functional heterogeneity is
associated with the cell cycle status of murine HSC, and that HSCs in
the G0/G1 phase of the cell cycle are more
enriched for radioprotection and long-term reconstitution activities
compared with cells in S/G2/M phase.37 To
evaluate this correlation between cell cycle status and long-term
repopulating activity, we measured the cycling status of HSC
populations isolated with Rh-123 or BodVer in the presence or absence
of MDR blockers. Cells isolated by BodVerlow or
Rh-123low staining were predominantly in the
G0/G1 phase, in either the presence or absence
of MDR modulators (Table 2). In addition, verapamil did not influence
the frequency of cycling cells in the BodVerhigh and
Rh-123high populations, while both cyclosporin A and
reserpine enhanced the staining of cycling cells with either Rh-123 or
BodVer. The engraftment results shown in Figs 4 and 6 suggest that the
G0/G1 cell cycle state by itself is a poor
marker for the subset of Thy-1.1lowSca-1+Lin HSC that
mediates long-term engraftment because equivalent enrichments for
G0/G1 cells were obtained with either probe
(Table 2).
Analysis of transmission electron micrographs has suggested a selective
increase in mitochondrial size, but not number, in Rh-123high HSC when compared with Rh-123low
cells.33 The reported twofold increase in mitochondrial
size would not by itself be sufficient to account for the 30-fold
increase in fluorescence intensity that is apparent between
Rh-123low and Rh-123high HSC (Fig 1). To
further evaluate the mitochondrial localization of Rh-123 in
Thy-1.1lowSca-1+Lin HSC, we
used laser scanning confocal microscopy to visualize the intracellular
distribution of the probe and correlated these observations with
transmission electron micrographs of parallel populations of cells
(Figs 7 and 8). These results confirm cellular localization of the
probe, which is coincident with mitochondrial distribution in both cell
populations and show a marked bipolar perinuclear clustering of
activated mitochondria in Rh-123high cells. This
observation may indicate segregation of mitochondria before cell
division, or may be a result of cellular activation. In contrast, the
distribution of fluorescence in the Rh-123low cells was
rather uniform in the majority of cells analyzed. If P-gp-mediated
efflux of Rh-123 was the sole mechanism by which these cell subsets are
resolved, one would expect to detect quantitatively distinct, but
morphologically equivalent, distribution of fluorescence in cells
separated by Rh-123 staining; this clearly was not the case (Fig 7).
Collectively, the results reported here support the hypothesis that
mitochondrial membrane charge potentials play a major mechanistic role
in the ability of Rh-123 to discriminate the quiescent long-term
repopulating HSC population from activated progenitor cells. Active
efflux of fluorescent probes by MDR mechanisms seems to exaggerate the
differential intensity between low and high Rh-123 staining,
particularly in older animals, but does not absolutely correlate with
long-term repopulating HSC function. Any influence of MDR in defining
primitive HSC would seem to be specific to the P-gp efflux pump, as
pump substrates with a broader substrate specificity range (BodVer and
NAO) do not replicate the ability of Rh-123 to identify primitive HSC
in young animals. This conclusion is strengthened by recent studies of
mice genetically deficient in both mdr1a and mdr1b38;
efflux of Rh-123 from bone marrow progenitor cells derived from the
double knockout animals is completely absent, but partially present in
either single knockout, proving a specific role for both of the
mdr1-type P-gps in determining the Rh-123 staining intensity of mouse
hematopoietic cells. However, no functional analysis of cell subsets
separated on the basis of Rh-123 staining intensities was reported, as
the mixed genetic background of these animals precludes transplantation
experiments. If mitochondrial activation plays a major role in the
mechanism of Rh-123 staining in HSC as suggested here, one would
predict that Rh-123 still will be capable of segregating functional
subsets of primitive hematopoietic cells in the double mdr knockouts.
This and other studies of the mechanism(s) underlying the ability of
Rh-123 to identify primitive HSC will help guide future efforts to
identify more specific probes for this population of cells,
particularly in situations where P-gp expression is coincident with HSC
function.8,26,35,39,40
The significance of MDR function in the HSC compartment remains to be
determined. The mdr1 double knockout animals display no physiologic
abnormalities in any parameter measured, including hematologic and
immunologic phenotype and function.38 However, vital
functions of these proteins may be supplemented by other membrane pumps
in their absence. Monoclonal antibodies against P-gp have been shown to
inhibit cytokine secretion from PHA-activated lymphocytes,41,42 suggesting that a possible physiologic
role of P-gp could be considered in hematopoiesis via hematopoietic growth factor secretion or regulation. Upregulation of P-gp function in
older animals may suggest a role in maintaining the stem cell pool with
age, a proposal that is supported by studies in which overexpression of
P-gp in transplanted bone marrow was associated with extended serial
transplantation potential.43 Although the results reported
in this study do not support a major role for MDR function in the HSC
compartment of young animals, further studies aimed at characterizing
this function during the course of aging are clearly warranted.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted January 26, 1998.
Supported by Grants No. RO1 HL56857 and P50 DK49219 from the National
Institutes of Health and the Huntsman Cancer Institute at the
University of Utah. The Flow Cytometry, Cell Imaging, and Irradiation
core facilities of the Huntsman Cancer Institute, supported by National
Cancer Institute Cancer Center Support Grant No. P30 CA42014, were used
for these studies.
Address reprint requests to Gerald J. Spangrude, PhD,
Department of Pathology, Room 5C 130 SOM, University of Utah, Salt Lake City, UT 84132.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Ann Cline and Diane Brooks for excellent technical
assistance in flow cytometry. Dr Wayne Green provided advice and
assistance in the cell cycle analysis studies. We also thank Dr Preet
Chaundry for helpful discussions, suggesting the experiments to test
efflux of fluorescent probes using MDR transfectant cell lines, and
reviewing the manuscript.
| |
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Abstract
Introduction
Abstract