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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3681-3687
Lymphohematopoietic Engraftment in Minimally Myeloablated Hosts
By
F.M. Stewart,
S. Zhong,
J. Wuu,
C.-c. Hsieh,
S.K. Nilsson, and
P.J. Quesenberry
From the Cancer Center and Division of Hematology-Oncology,
University of Massachusetts Medical Center, Worcester, MA.
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ABSTRACT |
The concept that myeloablation to open space was a prerequisite for
marrow stem cell engraftment has been challenged by studies showing
high rates of engraftment in nonmyeloablated mice (Stewart et al,
Blood 81:2566, 1993; Quesenberry et al, Blood Cells
20:97, 1994; Brecher et al, Blood Cells 5:237, 1979; Saxe et
al, Exp Hematol 12:277, 1984; and Wu et al, Exp Hematol
21:251, 1993). However, relatively large numbers of marrow
cells were necessary to achieve high long-term donor percentages. We
have demonstrated, using a BALB/c male/female marrow transplant model
and detecting male DNA in host tissues by Southern blot or fluorescent
in situ hybridization, that exposure to doses of irradiation that cause minimal myeloablation (50 to 100 cGy) leads to very high levels of
donor chimerism, such that relatively small numbers of marrow cells (10 to 40 million) can give donor chimerism in the 40% to 100% range.
Studies of radiation sensitivity of long-term engrafting cells have
shown that 100 cGy, although not myelotoxic, is stem cell toxic, and
indicate that the final host:donor ratios are determined by competition
between host and donor stem cells. These data indicate that low levels
of irradiation should be an effective approach to nontoxic marrow
transplantation in gene therapy or in attempts to create allochimerism
to treat such diseases as cancer, sickle cell anemia, or thalassemia.
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INTRODUCTION |
CLASSIC TRANSPLANTATION models involve
lethal myeloablation, a setting in which a few or very large numbers of
infused marrow stem cells will give 100% donor cells after
transplantation; in the case of low numbers of infused marrow cells,
hematopoiesis in the transplanted animal is oligoclonal,1,2
whereas with large numbers, polyclonal hematopoiesis
ensues.3 In addition, lower levels of irradiation have been
extensively evaluated for effects on marrow recovery, but not in a
quantitative fashion in syngeneic or autologous models, because most
studies have not used cells with a donor-specific marker.
We have previously described very high levels of stable marrow cell
engraftment in normal nonmyeloablated murine hosts, a model in which
donor cells are competed against host cells.4 Data on cell
dose effects suggested that donor cell engraftment was quantitative at
the stem cell level and that the final percentage of donor chimerism
might be determined by the ratio of host to donor stem cells, rather
than by any effect on opening space for stem cell
engraftment.5 Previous studies using a variety of stem/progenitor assays have indicated relatively high rates of killing
at levels of irradiation between 50 and 100 cGy.6,7 The
present data suggest that 40 million male BALB/c marrow cells with
quantitative stem cell engraftment into female BALB/c hosts exposed to
50 to 100 cGy total body irradiation, a level that might eliminate the
bulk of host stem cells (vide infra), should give high donor-host
ratios in marrow, spleen, and thymus.
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MATERIALS AND METHODS |
Mice.
Six- to 8-week-old BALB/c H-2D mice (Taconic Farm,
Germantown, NY) were housed in a conventional clean
facility for at least 1 week before experimental use. Mice received
mouse chow and acidified water ad libitum.
Marrow cell suspension.
Mice were killed by cervical dislocation and bone marrow collected from
tibia and femurs. Cells were flushed from femurs and tibia with cold
phosphate-buffered saline (PBS) supplemented with 5% heat-inactiviated
(HI) fetal calf serum (FCS; Hyclone, Logan, UT) and filtered through a
40-µm filter (Becton Dickinson, Franklin Lakes, NJ). The cells were
washed and suspended for injection in PBS.
Transplantation.
Normal or irradiated female BALB/c mice were infused with from 1 × 106 to 40 × 106 normal male
BALB/c marrow cells or 40 × 106 irradiated male
BALB/c marrow cells, and the percentage of male DNA per female was
determined by Southern blot analysis as described below.
Southern blot.
Spleen, thymus, and marrow cell extracts were prepared by lysis in 0.15 mol/L NaCl, 0.02 mol/L Tris, 0.02 mol/L EDTA, and 1% sodium dodecyl
sulfate followed by organic extraction with proteinase K
(Sigma, St Louis, MO), RNAse (Sigma), and
phenol-chloroform and precipitation in ethanol. The presence of
Y-specific DNA sequences was assessed using a pY2-cDNA probe (donated
by I. Lemischka, Princeton University, Princeton, NJ). DNA samples were
digested with the restriction enzyme Dra I and separated by gel
electrophoresis in 0.8% agarose (GIBCO, Richmond, CA) according to
established Southern blotting techniques. Sample loading was assessed
by reprobing membranes with a partial or full length cDNA for
interleukin-3 (donated by J. Ihle and DNAX, Palo Alto, CA). Probes were
32P-labeled using a random primed labeling kit (Boehringer
Mannheim, Mannheim, Germany) and autoradiographs were made using Kodak
XRP x-ray film (Eastman Kodak, Rochester, NY). Blots were exposed to
photostimulatable storage phosphorimaging plates, and the percentage of
male and female DNA was quantitated after scanning the plates with a
GS-363 molecular imager system (Bio-Rad, Hercules, CA).
Differential cell count.
Cells from spleen samples from posttransplant mice were centrifuged
onto slides (500 rpm for 5 minutes) by cytospin (Shandon, Pittsburgh,
PA). Slides were fixed in 100% methanol and stained with
Wright-Giemsa staining. At least 100 mononuclear cells were counted
using the Nikon E400 (MicroVideo Instruments, Avon, MA) with 40×
lens to determine lymphocyte percentages.
Fluorescence in situ hybridization (FISH).
Cytospins were fixed in a 50% Carnoys (75% methanol/25% acetic acid)
and 50% PBS solution for 10 minutes before being baked at 72°C for
1 hour. The slides were then further fixed in 100% Carnoys for 5 minutes before permeabilization using proteinase K (0.2 µg/mL)
enzymatic digestion in 2 mmol/L CaCl2 and 20 mmol/L Tris buffer (pH 7.5) at 37°C for 1.5 minutes.
The cytospins were dehydrated once more and hybridized with a
digoxigenin-labeled Y chromosome-specific probe at 45°C overnight.
Unbound probe was removed by stringent washings in 3 changes of 50%
formamide in 2× SSC and 2 changes of 2× SSC at 45°C.
After washing in 4× SSC (0.6 mol/L NaCl and 0.06 mol/L sodium
citrate, pH 6.4) at room temperature, the slides were bleached using a
blocking buffer consisting of 5% FCS, 5% nonfat milk (Shaw's,
Bridgewater, MA) and 0.05% Triton X-100 (Sigma) in 4× SSC for 15 minutes. Detection of digoxigenin was performed using anti-digoxigenin
rhodamine-labeled Fab fragments (Boehringer Mannheim) at a
concentration of 6.5 µg/mL in PBS containing 2% bovine serum albumin
(BSA) fraction V (Sigma) for 30 minutes in the dark. Nonbound antibody
was removed through three extensive, light-protected washings: first in
4× SSC for 10 minutes, then in 4× SSC containing 0.05%
Triton X-100 for 10 minutes, and finally in 4× SSC for 10 minutes. Cytospins were counterstained in 0.4 µmol DAP1
(4.6-Diamidino-2-phenylindole; Sigma) and mounted in the antifade media
Vectashield (Vector, Burlingame, CA). Specific positive
label was confirmed under the microscope by a visual check at
excitation and emission wavelength other than that of rhodamine.
FISH on sections of paraffin-embedded whole murine femur.
Sections of paraffin-embedded murine femurs were prepared as previously
noted.8 Mice were perfused with 4% paraformaldehyde into
the descending aorta and femurs were removed, decalcified in EDTA,
dehydrated in graded ethanol, and cleared in mineral spirits (Aldrich
Chemical Co, Milwaukee, WI) before infiltration and embedding in
Paraplast x-tra (Oxford Labware, St Louis, MO). Five-micrometer femoral
sections were mounted on 0.01% poly-L-lysine (molecular weight 150,000 to 300,000; Sigma Diagnostics, St Louis, MO) subbed
slides, deparaffinized in xylene, rehydrated in graded ethanol, and
washed in distilled water and then PBS. Binding to nonspecific proteins
was blocked using blocking buffer consisting of 5% FCS, 5% nonfat
milk (Shaw's) and 0.05% Triton X-100 (Sigma) in 4× SSC (0.06 mol/L NaCl and 0.06 mol/L sodium citrate, pH 6.4) for 15 minutes at room temperature. The FISH-labeled sections were mounted
using Vectashield (Vector), and each section was mounted in
approximately 5 µL of mounting media. The cover slips were sealed
with nail polish. Mounted slides were stored at 4°C protected from
light. Sections were evaluated using a Nikon Microphot-FXA microscope
equipped for epifluorescence (100-W high-pressure mercury lamp;
MicroVideo Instruments). Sections were viewed using a
40/1.0 oil Nikon objective. Fluorescent photomicrographs were taken
with Kodak Ektachrome EL IS0 400/27°C. UV excitation was performed to evaluate nonspecific autofluorescence not representing the male
signal.
Irradiation of cells or mice.
Balb/c female hosts, Balb/c male donors, or male Balb/c marrow cells
suspended in PBS in 50-mL conical tubes received varying doses of
irradiation from a cesium source (Gamma cell 40; Nordian, Kanata, Ontario, Canada) at a dose rate of 92 to 94 cGy/min.
Purification of stem cells.
Bone marrow was collected from femurs, tibia, and iliac crests by
grinding in PBS supplemented with 5% HI FCS (Hyclone) using a mortar
and pestle. The bone fragments were washed multiple times and the
supernatant cell suspension and wash fractions were filtered through a
40-µm filter (Becton Dickinson) to remove large bone particles. High
lipid concentrations were reduced by the centrifugation and
resuspension of the cells in fresh buffer. The cells were allowed to
sit on ice for 5 minutes so that small bone particles would settle out.
The cell supernatant, depleted of these fragments, was then diluted to
107 cells/mL PBS 5% HI FCS.
Lineage-negative cells were isolated in a manner very similar to that
previously described by Bertoncello et al.9 Briefly, low-density cells (<1.0777 g/mL) were isolated by discontinuous density centrifugation using Nycoprep for animals (Accurate Chemical and Scientific Corp, Westbury, NY), washed, and resuspended at 108 cells/mL PBS 5% HI FCS. Cells were labeled with a
cocktail of primary antibodies: anti-B220 (B cells)
Coffman10; anti-Mac-1 (macrophages)11;
anti-Gr-1 (neutrophils)12; anti-Lyt-2 (CD8) and
anti-L3T4 (CD4) (T cells) Cobbold13
(Becton Dickinson); YW25.12.7 (erythroid precursors)14; and
TER119 (erythrocytes; a kind gift from Tatsuo Kina, Chest Research Institute, Kyoto University, Kyoto, Japan). Each batch of
antibody was evaluated by flow cytometric analysis for the concentration that resulted in the greatest shift in mean channel fluorescence and/or the percentage of positive cells detected. The optimal dilution for each antibody was in the range of 1:10 to
1:100 (final). After 15 minutes of incubation on ice, the labeled cells
were washed in PBS 5% HI FCS and resuspended at 108
cells/mL PBS 5% HI FCS. The cells were incubated with sheep antirat IgG-conjugated immunomagnetic polystyrene spheres (M-450 Dynabeads; Dynal, Lake Success, NY) at 4°C for 3 minutes by adding a 1:1 bead:cell ratio. The beads were suspended in PBS 5% HI FCS and, when
added to the cells, resulted in 1.5 times the original cell volume.
Immunomagnetic bead-rosetted cells were removed using a magnetic
particle concentrator (Dynal MPC-6), and the unrosetted cells remaining
in suspension were obtained by pipette. The beads were washed three
times, and the supernatant was pooled with the cell suspension. The
unrosetted cells were further incubated with antirat-conjugated
immunomagnetic beads at 4°C for 20 minutes by adding a 4:1
bead:cell ratio, resulting in double the original cell volume. Again,
immunomagnetic bead-rosetted cells were removed using a magnetic
particle concentrator, and the unrosetted cells remaining in suspension
were harvested by pipette. The beads were washed three times, and the
supernatant was pooled with the cell suspension.
Rhodamine 123 (Rho)/Hoechst 33342 (Ho) labeled cell separation.
Immunomagnetically enriched cell suspensions were washed and
resuspended at 106 cells/mL in PBS 5% HI FCS and incubated
in a final Rho concentration 0.1 µg/mL for 20 minutes at 37°C in
the dark. A final Ho concentration of 10 µmol/L was then added, and
the cells were incubated at 37°C in the dark for 1 further hour.
The cells were washed in ice-cold PBS 5% HI FCS and held on ice in
preparation for sorting.
The overall procedure for sorting of Lin ,
Rholow/Holow cells from murine bone marrow is
essentially as described by Wolf et al.15 Briefly, this
procedure involved recovering lineage-depleted bone marrow cells as
described above and labeling them with Rhodamine-123 and Hoechst-33342.
The labeled cells were then sorted on MoFlo (Cytomation, Fort Collins,
CO) cell sorter to recover the lowest 10% Rhodamine-123 staining and
lowest 3% Hoechst-33342 staining cells.
Assessment of cell cycle status of engrafting stem cell.
Donor male mice were injected with 900 mg/kg hydroxyurea intravenously.
Control animals received PBS intravenously. Marrow cells were harvested
3 hours after the injection, and cells were pooled separately for
hydroxyurea group and control group. Forty million cells from
hydroxyurea-treated mice were infused into each of 10 female recipients
that were exposed to 100 cGy 4 hours previously. Forty million
PBS-treated bone marrow cells were infused into each of 10 female
recipients exposed to 100 cGy 4 hours previously. Mice were killed and
analyzed 8 weeks posttransplantation.
Assessment of hematologic effects of minimal irradiation.
To determine the hematologic effects of low doses of irradiation on the
normal host, animals in groups of five received low doses of whole
animal irradiation (0, 20, 50, and 100 cGy) without donor cells. At
days 2, 7, and 14, animals were killed and tissues were analyzed for
white blood cell count (×103/µL), platelet count
(×109/L), and marrow cellularity
(×103; 2 tibias and 1 femur).
Statistical methods.
The nonparametric Kruskal-Wallis one-way rank test was used for all
statistical comparisons.16 Kruskal-Wallis analysis and Cuzick two-sided test were used to evaluate engraftment trends over
time.
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RESULTS |
Engraftment in minimally irradiated hosts.
To determine if low levels of irradiation might alter competitive
pressure after engraftment to favor donor cells, BALB/c female mice
were treated with 0, 10, 20, 50, or 100 cGy; were infused with 40 million normal BALB/c male marrow cells; and were evaluated at 2, 5, and 8 months after marrow infusion (Figs 1 and 2). Engraftment was quantitated by
determining the percentage of male DNA in host marrow, spleen, and
thymus using Southern blot analysis and the Y-specific pY2 DNA probe.
These results indicate very high rates of male cell engraftment at 100 cGy with significant, but lower levels of engraftment seen at both 20 and 50 cGy. Engraftment was stable from 2 to 8 months after marrow infusion and in general was comparable in all three tissues analyzed. Increasing the dose of irradiation to 200 to 300 cGy did not increase engraftment at 2 months (data not shown). The high rates of engraftment seen with Southern blot analysis were confirmed with FISH on marrow sections (Fig 3).
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| Fig 1.
Effect of minimal irradiation on engraftment. Balb/c
female hosts received graded doses of whole animal irradiation (0, 10, 20, 50, and 100 cGy) followed 4 hours later by infusion of 40 × 106 normal Balb/c male marrow cells of the same strain in a
single injection. To evaluate repopulation patterns by Southern blot analysis, we used the Y-chromosome specific pY-2 probe.
Female recipient mice were killed 2 months after 0, 10, 20, 50, and 100 cGy and infusion of male marrow cells with 5 to 10 transplanted animals
per dose level. A dose of 100 compared with 0 cGy showed increased
engraftment in marrow, spleen, and thymus (P < .01). At 20 and 50 cGy, both bone marrow and spleen engraftment was increased
compared with 0 cGy (P < .02). Compared with 50 cGy, 100 cGy
produced superior engraftment in spleen, thymus, and marrow (P < .01).
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| Fig 2.
Female recipient mice were killed at 2, 5, and 8 months
after 0, 10, 50, 100 cGy. (A) Male marrow engraftment over time. (B) Southern blot showing 2-month time point. Kruskal-Wallis analysis and
Cuzick two-sided test for trend over time showed that engraftment was
sustained (eg, no downward trend) for bone marrow and spleen (not
shown). Thymic engraftment (not shown) showed a modest decrease over
time (P < .05 for all levels of irradiation).
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| Fig 3.
In situ hybridization of femoral marrow sections. (a)
Balb/c male control showing Y-chromosome specific signal (arrows). (b) Balb/c female control showing Y-chromosome negative cells. (c) Balb/c
female transplant recipient 6 months after receiving 100 cGy and 40 × 106 Balb/c male marrow cells. (d) Same as (c) except
excited with UV. The transplant resulted in 81% marrow chimerism as
determined by Southern blot analysis. Arrows indicated positive marrow
cells of donor origin. Scale bar for (a) through (d) = 30 µm.
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Engraftment was present in myeloid lineages, as shown by the high
percentage of chimerism seen in marrow, which is predominantly myeloid. Engraftment was also represented in lymphoid
lineages, as shown by the high chimerism rates in spleen, which has
greater than 90% lymphocytes (Table 1).
Doses of 50 to 100 cGy induced moderate and transient depression of
peripheral white blood cells, platelets, and marrow cellularity (Fig 4) and no apparent toxicity.
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| Fig 4.
Effect of minimal irradiation on differentiated cell
levels. To determine the hematologic effects of low doses of
irradiation on the normal host, animals in groups of five received low
doses of whole animal irradiation (0, 20, 50, and 100 cGy) without
donor cells. At days 2, 7, and 14, animals were killed and tissues were analyzed for white blood cell count (×103/µL), platelet
count (×109/L), and marrow cellularity
(×103; 2 tibias and 1 femur).
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Effect of cell dose on engraftment in nonablated, minimally ablated,
and intensively ablated hosts.
Cell dose appears to be a critical factor in determining the levels of
engraftment. We assessed this effect by infusing 2, 10, or 40 million
male BALB/c marrow cells into nonirradiated BALB/c female hosts or into
female hosts exposed to 100 cGy or 700 cGy before marrow infusion
(Fig 5). At 700 cGy, full repopulation (100% donor) was seen with 2 million cells, whereas this level of
engraftment was approached with 40 million donor cells in mice exposed
to 100 cGy. Ten million cells gave approximately 40% engraftment at 2 months. Although exact extrapolation is difficult, this relative level
of cells (10 million) is probably less than the average human marrow
harvest used for therapeutic transplantation and indicates that minimal
myeloablation (100 cGy) may be feasible for clinical marrow
transplantation.
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| Fig 5.
Cell dose and engraftment in minimally irradiated
recipients. Male marrow cells in doses of 2, 10, or 40 million cells
were infused into female recipients treated with 0, 100, and 700 cGy, and the percentage of engraftment was determined after 2 months. The
results show that donor cell readout in hosts after transplant is
related to cell dose and irradiation dose to recipient animals. Cell
doses as low as 10 million may yield up to 40% in the readout in
animals receiving 100 cGy. Experimental points include 5 mice per cell
level; where standard errors are not apparent, they fall within the
symbol.
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Engraftment of purified stem cells in minimally ablated hosts.
To establish that the hematopoiesis observed at 2 months and beyond in
fact was derived from stem cells and not from long-lived differentiated
cell populations, such as lymphocytes or monocytes, we evaluated the
capacity of marrow depleted of differentiated lineages
(lin) to engraft (Table
2). We also evaluated highly purified stem cells separated on the basis
of low Hoechst and Rhodamine (Hoechstlow
Rhodaminelow) staining for their capacity to engraft in
mice exposed to 100 cGy. These data show high levels of engraftment at
100 cGy, indicating that stem cells are in fact being monitored.
Engraftment levels were lower than expected from the starting number of
cells. The explanations for this could include loss of cells with
purification, depletion of a facilitator cell population, or high rates
of progenitor engraftment in the unseparated marrow.
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Table 2.
Engraftment of Separated/Selected
Cells Into Minimally Irradiated Hosts: Lineage-Depleted Male
Marrow Cells Into Female Hosts
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Cell cycle status of engrafting stem cell.
The cells engrafting into minimally myeloablated mice are quiescent as
determined by administration of hydroxyruea or PBS to donor mice 2 hours before death (Fig 6).
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| Fig 6.
Effect of hydroxyurea on donor stem cell engraftment in
hosts irradiated with 100 cGy. To selectively kill cells transiting S
phase, donor male BALB/c mice received an injection of 900 mg/kg hydroxyurea 3 hours before death. Mice injected with PBS constituted the control donor group. The two cell populations were infused into
separate groups of irradiated (100 cGy) female hosts and the percentage
of male cells in female host marrow was assessed 8 weeks after cellular
infusion. Actively cycling cells will be killed by hydroxyurea and
noncycling cells will be spared. The present results are from two
experiments with 5 mice per experiment and show no significant
differences, indicating that the engrafting cells were quiescent.
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Stem cell survival and repopulating ability after exposure to minimal
irradiation in vivo or in vitro.
We examined the effect of in vitro or in vivo exposure to 0, 20, 50, 70, 100, or 200 cGy on the ability of 40 million BALB/c marrow cells to
engraft and populate irradiated (100 cGy) female BALB/c hosts 2 months
postinfusion (Fig 7). Exposure to 100 cGy, either in vitro or in vivo, reduced the populating ability to 14% of
that seen with marrow that had not been exposed to irradiation.
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| Fig 7.
Effect of minimal irradiation on stem cells irradiated in
vivo or in vitro. Male marrow cells from BALB/c male mice were exposed to irradiation (0, 20, 50, 70, 100, and 200 cGy) in vitro and 40 × 106 was immediately infused into female recipients
irradiated with 100 cGy. In another experiment, BALB/c male mice in
groups of 5 were exposed to irradiation (0, 20, 50, 70, and 100 cGy),
marrow cells were harvested, and 40 × 106 male marrow
cells were immediately infused into female recipients irradiated with
100 cGy (groups of 5). At 2 months, the percentage of male engraftment
was determined. The results show that increasing doses of minimal
irradiation have a profoundly toxic effect on stem cells irradiated in
vitro or in vivo.
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DISCUSSION |
Previous dogma suggested that irradiation facilitated engraftment by
opening space. As noted above, the studies of engraftment in
nonmyeloablated hosts have challenged this concept. Only a few studies
have compared engraftment of marrow stem cells in mice subjected to low
dose irradiation with that seen in mice receiving no irradiation. These
studies used higher levels of irradiation or showed only minimal
engraftment.17,18 Shirot and Tavassoli19
evaluated mice treated with 100 to 2,000 cGy and studied
perfusion-fixed marrow by transmission electron microscopy. They showed
sloughing and denudation of plasma membranes on the luminal side of the
endothelium in irradiated mice. Other changes, predominately noted with
doses of greater than 250 cGy, included membrane vesiculation,
dilatation of the perinuclear space and rough endoplasmic reticulum,
focal cytoplasmic swelling of endothelium, and bleeding into the marrow
space. Although injury was apparent in mice exposed to 100 cGy, this
appeared to heal quickly. At 1,000 cGy, cellular disruption was
irreversible. Work by Hendrikx et al20 using
purified stem cells labeled with the fluorescent membrane stain PKH-26
and engrafted into lethally irradiated mice or nonirradiated mice
indicated that homing to nonirradiated marrow was 2.5 times higher than
to irradiated bone marrow, suggesting a deleterious effect on
engraftment of stem cells by high levels of host irradiation.
Although the low levels of irradiation used here could be physically
opening space, the minimal effect on total marrow cellularity, the high
levels of stem cell engraftment seen in nonmyeloablated mice,21-24 and the above-noted homing studies all argue
against this mechanism. Our previous data suggest that the final
host:donor ratios might be determined by competition between host and
donor stem cells. To test this directly, we examined the effect of in vitro or in vivo exposure to 0, 20, 50, 70, 100, or 200 cGy on the
ability of 40 million BALB/c marrow cells to engraft and populate irradiated (100 cGy) female BALB/c hosts 2 months postinfusion (Fig 6).
Exposure to 100 cGy, either in vitro or in vivo, reduced populating
ability to 14% of that seen with marrow that had not been exposed to
irradiation. This level of host stem cell reduction, either by death or
alteration in function, coupled with the infusion of 40 million marrow
cells (in a host presumed to have approximately 300 million total
marrow cells), could explain the high levels of donor cell chimerism on
a simple stem cell competition model (Fig
8). These data indicated that engraftment at the stem cell level is
very efficient in minimally myeloablated mice and that the final
donor:host ratios are determined by competition between host and donor
stem cells. It is possible that the majority of donor cells are
radiosensitive but that there is a population existing of relatively
radioresistant cells. Minimal myeloablation, in concert with clinically
obtainable stem cell numbers, provides an approach to gene therapy or
for the creation of allochimeras in such diseases as sickle cell anemia
or thalassemia, which should be both effective and nontoxic. In
addition, this approach, with appropriate immunosuppression, may allow
for the creation of allochimeras in cellular immunotherapy strategies
for the treatment of cancer.
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| Fig 8.
Model of competitive stem cell engraftment into (A) normal
(nonirradiated host) and (B) minimally irradiated host (100 cGy).
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FOOTNOTES |
Submitted August 27, 1997;
accepted January 12, 1998.
P.J.Q. was supported by grants from the National Institutes of Diabetes
and Digestive and Kidney Diseases (P0-1 DK 50222-01, R0-1 DK 49650-03, and R0-1 DK 27424-15) and the National Heart, Lung and Blood Institute
(P0-1 HL 56920-01). F.M.S. was supported by a grant from the Leukemia
Society of America (New York, NY; Grant No. 6207). J.W. and C.-c.H.
were supported in part by a grant from the Howard Hughes Medical
Institute (HHMI Grant No. 76296-550401).
Address reprint requests to F.M. Stewart, MD, Division of
Hematology/Oncology, University of Massachusetts Medical Center, 55 Lake Ave N, Worcester, MA 01655.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
REFERENCES |
1.
Mintz B,
Anthony K,
Litman S:
Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts.
Proc Natl Acad Sci USA
81:7835,
1984[Abstract/Free Full Text]
2.
Lemischka IR,
Raulet D,
Mulligan RC:
Developmental potential and dynamic behavior of hematopoietic stem cells.
Cell
45:917,
1986[Medline]
[Order article via Infotrieve]
3.
Harrison DE,
Clinton MA,
Lerner C:
Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells.
Proc Natl Acad Sci USA
85:822,
1988[Abstract/Free Full Text]
4.
Stewart FM,
Crittenden R,
Lowry PA,
Pearson-White S,
Quesenberry PJ:
Long-term engraftment of normal and post-5-Fluorouracil murine marrow into normal myeloablated mice.
Blood
81:2566,
1993[Abstract/Free Full Text]
5.
Rao SS,
Peters SO,
Crittenden RB,
Stewart FM,
Ramshaw HS,
Quesenberry PJ:
Stem cell transplantation in the normal nonmyeloablated host: Relationship between cell dose, schedule and engraftment.
Exp Hematol
25:114,
1997[Medline]
[Order article via Infotrieve]
6.
Schoeters GE,
Vanderboroght OL:
Haemopoietic stem cell concentration and CFUs in DNA synthesis in bone marrow from different bone regions.
Experientia
36:459,
1980[Medline]
[Order article via Infotrieve]
7. Hall EJ: Radiobiology for the Radiologist. Philadelphia, PA,
Lippincott, 1988
8.
Nilsson SK,
Hulspas R,
Heinz-Ulrich GW,
Quesenberry P:
In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femurs.
J Histochem Cytochem
44:1069,
1996[Abstract]
9.
Bertoncello I,
Bradley TR,
Watt SM:
An improved negative immmunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.
Exp Hematol
19:95,
1991[Medline]
[Order article via Infotrieve]
10.
Coffman RL:
Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B cell development.
Immunol Rev
69:5,
1982[Medline]
[Order article via Infotrieve]
11.
Springer T,
Galfre G,
Secher DS,
Milstein C:
MAC-1: A macrophage differentiation antigen identified by monoclonal antibody.
Eur J Immunol
9:301,
1979[Medline]
[Order article via Infotrieve]
12.
Hestdal K,
Ruscetti FW,
Ihle JN,
Jacobsen SE,
Dubois CM,
Kopp WC,
Longo DL,
Keller JR:
Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells.
J Immunol
147:22,
1991[Abstract]
13.
Cobbold SP,
Jayasuriya A,
Nash A,
Prospero TD,
Waldmann H:
Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo.
Nature
312:548,
1984[Medline]
[Order article via Infotrieve]
14.
Watt SM,
Gilmore DJ,
Davis JM,
Clark MR,
Waldmann H:
Cell surface makers on haemopoietic precursors. Reagents for the isolation and analysis of progenitor cell subpopulations.
Mol Cell Probes
1:297,
1987[Medline]
[Order article via Infotrieve]
15.
Wolf NS,
Kone A,
Priestley GV,
Bartelmez SH:
In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection.
Exp Hematol
21:614,
1993[Medline]
[Order article via Infotrieve]
16. Armitage P, Berry G: Statistical Methods in Medical Research.
Oxford, UK, Blackwell Scientific, 1994
17.
Mardiney M III,
Malech HL:
Enhanced engraftment of hematopoietic progenitor cells in mice treated with granulocyte colony-stimulating factor before low-dose irradiation implications for gene therapy.
Blood
87:4049,
1996[Abstract/Free Full Text]
18.
van Os R,
Thames HD,
Konings AWT,
Down JD:
Radiation dose-fractionation and dose-rate relationships for long-term repopulating hemopoietic stem cells in a murine bone marrow transplant model.
Radiat Res
136:118,
1993[Medline]
[Order article via Infotrieve]
19.
Shirota T,
Tavassoli M:
Alterations of bone marrow sinus endothelium induced by ionizing irradiation: Implications in the homing of intravenously transplanted marrow.
Blood Cells
18:197,
1992[Medline]
[Order article via Infotrieve]
20.
Hendrikx PJ,
Martens CM,
Hagenbeek A,
Keij JF,
Visser JW:
Homing of fluorescently labeled murine hematopoietic stem cells.
Exp Hematol
24:129,
1996[Medline]
[Order article via Infotrieve]
21.
Quesenberry PJ,
Crittenden RB,
Lowry P,
Kittler EW,
Rao S,
Peters S,
Ramshaw H,
Stewart FM:
In vitro and in vivo studies of stromal niches.
Blood Cells
20:97,
1994[Medline]
[Order article via Infotrieve]
22.
Brecher G,
Tjio JH,
Haley JE,
Narla J,
Beal SL:
Transplantation of murine bone marrow without prior host irradiation.
Blood Cells
5:237,
1979[Medline]
[Order article via Infotrieve]
23.
Saxe D,
Boggs SS,
Boggs DR:
Transplantation of chromosomally marked syngeneic marrow cells into mice not subjected to hematopoietic stem cell depletion.
Exp Hematol
12:277,
1984[Medline]
[Order article via Infotrieve]
24.
Wu D,
Keating A:
Hematopoietic stem cells engraft in untreated transplant recipients.
Exp Hematol
21:251,
1993[Medline]
[Order article via Infotrieve]
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|
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Abstract
Introduction
Abstract