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Engraftment of Bone Marrow Cells Into Normal Unprepared Hosts:
Effects of 5-Fluorouracil and Cell Cycle Status
By Hayley S. Ramshaw, Sudhir S. Rao, Rowena B. Crittenden, Stefan 0. Peters, Heinz-Ulrich Weier,
and Peter J. Quesenberry
Bone marrow fromanimals treated with 5-fluorouracil (5FU)
competes equally with normal marrow when assessed in
vivo in an irradiated mouse, but shows markedly defective
engraftment when transplanted into noncytoablated hosts.
Using Southern Blot analysis and a Y-chromosome specific
probe, we determined the level of engraftment of male donor cells in the bone marrow, spleen, and thymus of unprepared female hosts. We have confirmed the defective engraftment of marrowharvested 6 days after 5FU (FU-6)and
transplanted into unprepared hosts and shown that this
defect is transient; by 35 days after 5FU (FU-35). engraftment
has returned t o levels seen with normal marrow. FU-6 marrow represents an actively cycling population of stem cells,
and we hypothesize that the cycle status of the stem cell
may relate t o its capacity t o engraft in the nonirradiated
host. Accordingly, we have evaluated the cycle status of
engrafting normal and FU-6 marrow intonormal hosts using
an in vivo hydroxyurea technique. We have shown that
those cells engrafting from normal marrow and over 70%
of the cells engrafting from FU-6 marrow were quiescent,
demonstrating no killing with hydroxyurea. We have also
used fluorescent in situ hybridization (FISH) analysis with a
Y-chromosome probe and demonstrated that normal and
postdFU engraftment patterns in peripheral blood were
similar t o those seen in bone marrow, spleen, and thymus.
Altogether these data indicate that cells engrafting in normal, unprepared hosts are dormant, and the defect that occurs after 5FU is concomitant with the induction of these
cells t o transit the cell cycle.
0 1995 by The American Societyof Hematology.
M
over time (1 to 25 months) and, using Southern Blot analysis
to detect Y-specific sequences in female hosts, engraftment
ranged from 15% to 42% at 21 to 25 months posttransplant.
Using a somewhat different approach, Wu and Keating6also
showed that a single injection of 20 X lo6 cells gives stable
engraftment at 8 weeks posttransplant, but the engrafted cells
show little tendency to differentiate.
Stewart et a15 also analyzed the capacity of BALBlc marrow (two tibiaeltwo femurs) harvested 6 days after 150 mg/
kg 5-fluorouracil (5FU) to engraft and persist in uninadiated
hosts. In their experiments the percentages of engrafted male
cells in the marrow ranged from 23% to 78% for recipients
of normal marrow, while recipients of post-5FU marrow had
from 0.1% to 39% cells carrying male DNA. These results
were surprising, considering studies showing that murine
marrow procured after 5FU is enriched for day 14 colonyforming unit-spleen-(CFU-S), high proliferative potential
colony-fodng cells (HPP-CFC), andmarrow
renewal
and competes equally withnormalmarrowwhen
transplanted into irradiated hosts in a competitive stem cell
assay.”.” This cycle induction effect of 5FU has been used
in mice, primates, and humans to facilitate retroviral integration in different gene therapy ~trategies.””~
It hasto be noted
that engraftment studies in irradiated mice were carried out
using C57BLl6 strain, and it maybe that thereis some
strain variation with the effects of 5FU, a possibility that is
currently being investigated.
The induction of a stem cell engraftment defect in nonirradiated hosts by treatment of donor mice with 5FU could
represent permanent damage to hematopoietic stem cells or
transient alterations in the functional state of the stem cell
that impairs engraftment. The 5FU treatment induces stem
cell cycling andan engraftment defect when transplanted
into noncytoablated host mice. This, coupled with the observationthat cytokine induction of stem cell cycling also
causes an engraftment defect in the unprepared host,” suggests thatwhen
primitive marrow stem cells progress
through cell cycle, they transiently lose the ability to engraft.
Such a phenomenon could explain the poor engraftment seen
with post-5FU marrow in normal hosts. In this report, we
URINE BONE MARROW transplantation and in vivo
stem cell assays have traditionally been into marrow
ablated hosts. Conventional dogma has held that spaces or
niches needed to be created by cytoablative therapy with
irradiation or cytotoxic drugs for marrow stem cells to home
and engraft.’ However, Micklem et al’, in 1968, demonstrated that engraftment into nonirradiated hosts was feasible
obtaining up to 8.5% T6T6 donor cells in CBA host marrow
at 3 months after transplantation of 20 million marrow cells
from normal CBA-T6T6 donors. These data were extended
by Brecher et a13 and Saxe et a14 who both were able to
demonstrate successful engraftment of murine bone marrow
into untreated hosts by using relatively high numbers of cells
for the transplants. Brecher et a1 showed that transplantation
of 40 million cells per day for 5 days into noncytoablated
hosts resulted in engraftment ranging from 16% to 25% at
2 to 13 weeks after the last transfusion. In a similar vein,
Saxe et a1 using chromosomally marked syngeneic marrow
cells into normal mice determined that donor cells in marrow
or spleen at 1 to 6 months posttransplantation ranged from
0% to 16%: Stewart et a15extended these data showing high
levels of persistent engraftment (over 2 years) in thymus,
spleen, and bone marrowwhen 40 million male BALBlc
cells were injected daily for 5 consecutive days into female
unirradiated BALBlc hosts. Engraftment appeared stable
From the Cancer Center, University of Massachusetts Medical
Center, Worcester, MA; the Department of Hematology/Oncology,
University of Virginia, Charlottesville, VA; and Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, CA.
Submitted November 14, 1994; accepted March IO, 1995.
Supported by National Institutes of Health Grant No.
POI.CA27466 and American Cancer Society Grant No. DHP-96.
Address reprint requests to Hayley S. Ramshaw, DPhil, UMMC
Cancer Center, 373Plantation St, Suite 207, Worcester, MA 01605.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8603-0013$3.00/0
924
Blood, Vol 86, NO3 (August l), 1995: pp 924-929
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925
ENGRAFTMENT OF CYCLING MARROW CELLS
have investigated the duration of the post-SFU engraftment
defect and the cell cycle status of normaland post-5FW
marrow cells engrafting into normal unirradiated hosts, with
the underlying hypothesis that engraftment potential of hematopoietic renewel cells in the unprepared mouse is linked
to the cell cycle status of these cells.
MATERIALS AND METHODS
Mice. BALBlc mice were purchased from Charles River Laboratories, Wilmington, MA and were maintained in virus-free conditions
at the Animal Facility at the University of Massachusetts Medical
Center, Biotech I1 building. All animals were given food and acidified water ad libitum. Mice were purchased at either 16 to 18 g or
18 to 22 g and were housed for a minimum of l week before
experimental use.
Transplantation of murine marrow from SFU-treated donors
into
normal untreated hosts. BALBlc female mice were transplanted
for 5 consecutive days with intravenous injections of cells from two
tibiae and two femurs from male BALBlc mice or with phosphatebuffered saline (PBS) (GIBCO-BRL, Bethesda, MD). Donor male
mice were untreated or injected via the tail vein with 5FU (150 mgl
kg) (SoloPak Laboratories, Franklin Park, IL) on day 0, then bone
marrow was procured 6 or 35 days after treatment (W-6 and FU35, respectively). Volumes for injections were 0.5 mL per transfusion. The percentage of male marrow in total host marrow cells was
determined using Southem blot analysis with the Y-chromosomespecific pY2 probe (a gift from Dr Ihor Lemischka, Princeton University, Princeton, NJ).I6Mice were killed at 7 or 10 weeks posttransplant. Southern blot analysis was also used to assess the percentage
of male marrow in spleen and thymus tissues. Fluorescent in situ
hybridization (FISH) was used to quantitate engraftment on a single
cell basis in peripheral blood.
Transplantation of murine marrow from hydroxyurea-treateddonors into normal hosts. BALBk female mice were transplanted
intravenously for 5 consecutive days with two tibiae and femurs
from male BALBlc mice that had been treated with hydroxyurea
(900 mgkg) (Sigma Chemical CO, St Louis, MO) or saline 2 hours
before killing.” Control recipient female mice received 0.5 mL PBS
(GIBCO-BRL) for 5 days. Percentage male cells in bone marrow,
spleen, and thymus was analyzed at 12 weeks posttransplant by
Southern blotting.
BALBlc female mice were also transplanted via tail vein injection
for 5 consecutive days with cells from two tibiae and two femurs
from male BALB/c mice that had been treated with 5FU (150 mgl
kg) (SoloPak Laboratories) on day 0 and either hydroxyurea (Sigma)
or saline on day 6. Donor mice, FU-6, were given hydroxyurea (900
mg/kg) or saline 2 hours before killing. Control recipient female
mice received 0.5 mLPBS (GIBCO-BRL). Recipient mice were
killed and DNA extracted from bone marrow, spleen, and thymus
for analysis at 6 weeks posttransplant.
Southern blot analysis. DNA extracts of murine bone marrow,
spleen, and thymus were prepared as described previously.’ For each
DNA sample, 5 pg was digested with the restriction enzyme Dra1 (Boehringer Mannheim, Indianapolis, IN) and separated by gel
electrophoresis in 0.8% agarose (GIBCO-BRL). DNA fragments
were transferred onto Zetaprobe nylon membranes (BioRad, Richmond, CA) by capillary transfer overnight in 0.4 N sodium hydroxide. The pY2 probe was labeled using a random prime labeling kit
(Boehringer Mannheim), and the blot was probed overnight at 65°C.
Autoradiography was performed for varying time intervals. Calculations of percentage male DNAin the samples were adjusted for
loading using an interleukin-3 (IL-3) cDNA probe (courtesy of J.
Ihle and DNAX, Palo Alto, CA). Percentage engraftment of male
cells in the female mice were quantitated using phosphorimage analysis (Molecular Dynamics Inc, Sunnyvale, CA.)
FISH. Peripheral blood was taken from transplant recipients by
cardiac puncture. Leukocytes were purifiedusing the Easy-Lyse
Whole Blood Erythrocyte Lysing kit (Leinco Technologies Inc, Ballwin, MO) and were fixed in Camoys solution (75% methanoll25%
acetic acid) before being dropped onto microscope slides for FISH
analysis. Slides were treated with proteinase K (0.2 pghnL) (Sigma
Chemical CO) in 2 mmol/L Tris buffer, at37°Cand
dehydrated
through an ethanol series. The DNAwas denatured in 70% formamide (GIBCO BRL) at 7 0 T , slides dehydrated once more, and
probe was applied immediately. The probe used in these studies was
a digoxygenin-labeled Y chromosome painting probe.I8 The probe
was incubated on the slides at 37°C overnight. Slides were washed
extensively in 50% formamide, 2X SSC followed by 4X SSC and
then were stained with rhodamine-antidigoxygenin antibodies
(Boehringer Mannheim). Slides were washedwith 4 X SSC and
cells were mounted in antifade solution containing 0.14 mglmL pphenylenediamine (Sigma) in 90% glycerol (Fisher Scientific, Pittsburgh, PA) and 0.4 pmol/L DAPI (4,6-Diamidino-2-phenylindole)
(Sigma). Positive cells were scored by visual counting against a
male and female control. Greater than 98 of every 100 male cells
stained positive, and the frequency of false positives was below 1
in 100 female cells (see Pallavicini et al” for a discussion of FISH
sensitivity).
Statistical analysis. Statistical analysis was performed using
Wilcoxon’s matched pairs test. Each experiment shows an individual
mouse for each data point, and within an experiment mice were
randomly paired for statistical analysis. The results were considered
significant when P S .05.
RESULTS
In the present study, we have administered 5FU (150 mg/
kg) to BALB/c mice, killed them 6 (FU-6) or 35 (FU-35)
days later and compared the capacity of these marrow cells
to engraft in host marrow, spleen, or thymus with marrow
cells from mice not treated with 5FU. In transplanted female
mice analyzed at 10 weeks posttransplantation, there was
a significant decrease in engraftment at 6 days post-SFU,
confirming previous reports’ (Fig 1 and Table 1). However,
in marrow and thymus, engraftment had returned to control
values 35 days after 5Fu. Recovery of engraftment in splenic
tissue appeared incomplete, consistent with our initial observations thatmarrowand
thymic engraftment correlated
closely, but splenic engraftment did not correlate well with
thymic or marrow engraftment. These data were confirmed
in a separate experiment in which engraftment was evaluated
7 weeks after marrow infusion. The results at 7 and 10 weeks
posttransplantation were combined and are presented in Table l and represent the analysis of 16 to 20 individually
transplanted mice. The differences between control and FU6 or FU-6 and FU-35 marrow recipients for marrow, spleen,
and thymus were significant at P values c.02 (W-6v normal) and <.05 (FU-6 v FU-35). These data indicate that
5FU administration to donor mice results in a transient engraftment defect into nonirradiated mice that recovers over
time.
We have previously shown that engraftment with normal
or post-SFU marrow is similar in T cells, B cells, and myeloid cells.’ We have now applied FISH to analyze engraftment levels in the peripheral blood of mice killed 7
weeks after transplantation with normal, FU-6, or FU-35
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926
RAMSHAW ET AL
Bone Marrow
No&
1
% Engraftment
2
3235
e donors
d
3
4
5
6
7
2R
23
25
29
18
Average f standard crmc = 27 f 2
N-6donors
% Engnlbnent
1
2
3
21
5
I
4
1
2
5
7
6
9
7
8 9
1 0 9
5
Averagefstnndardermr=9fZ
FU-35 donors
% Engraftment
1
2
3
36
S7 33
4
5
42 2234
6
7
8
9
16
17
16
Averagefstnndudemr=3OfS
Fig 1. Percentageof maleDNA in bone marrow of individual
recipient female mice given repetitive transplants normal,
of
FU-6, or FU35 bone marrow.Female mice received cells equivalent t o t w tibiae
o
and t w o femurs intravenously for
5 days and were killed 10
atweeks
posttransplantation. Southern blots were probedwith pY2 and the
percentage engraftment wascalculated using phosphorimageanalysis, taking a male control as 100% and a female control as 0%. All
samples were corrected for loading with an IL-3 probe. Statistical
analysis shows that the
differences between FU-6 and normal recipients are significant ( P i .02) and also for FU-6 against FU-35 (P i
.05).
marrow. These results parallel those presented above: 20%
2 3% positive cells in control (646 cells counted from two
mice), 7% -C 2% in FU-6 (157 cells counted from two mice)
and 18% 2 3% in FU-35 mice (475 cells counted from two
mice). This is the first demonstration of engraftment into
peripheral blood in unprepared hosts and shows that marrow
engraftment is mirrored by the presence of engrafted cells
in the peripheral blood.
Post-SFU marrow stem cells are initially induced into activecell cycle at the same timethat the above noted engraftment defect occurs. Thus, the cell cycle status of the
stem cell may be related to its capacity to engraft and persist
in vivo. Accordingly, we have assessed the cell cycle status
of the hematopoietic stem cells from normal mice that engraft into nonirradiated hosts. Hydroxyurea selectively kills
cells in S phase, and this approach gives essentially identical
results to those seen with in vitro high specific activity triti-
ated thymidine.” We have assessed the effects of hydroxyurea (900 mgkg), given 2 hoursbefore killing, onmale
BALBk donor cell engraftment into marrow, spleen, and
thymus of unprepared female BALBk hosts 12 weeks after
five consecutive infusions (two tibiaekwo femurs). Engraftment of the donor marrow cells from the hydroxyureatreated donors was compared with engraftment of donor
marrow cells from PBS-treated mice (Fig 2, Table 2). The
engraftment difference between the hydroxyurea and PBS
groups gives an estimate of the number of engrafting cells
that are actively proliferating, ie, transiting cell cycle. There
was no effect of hydroxyurea on the number of male cells
engrafting into female marrow, spleen, or thymus, indicating
that the cells, which engraft in this model, are noncycling,
quiescent stem cells.
5FU pretreatment induces cycling of stem cells assayed
in irradiated hosts.” If cycle status of stem cells is a determinant of engraftment and explains the poor engraftment seen
after SFU, then it would be anticipated that the majority of
engrafting cells in FU-6 marrow would notbe cycling; ie,
the cycling stem cells will have failed to engraft. We thus
used hydroxyurea (or PBS) treatment of FU-6 donors to
determine whether the FU-6 marrow cells engraftinginto
marrow, spleen, or thymus were proliferating or quiescent
(Table 3). In these studies, therewas a lowrate of engraftment with the FU-6 marrow and 73% to 75% of engrafting male cells in female marrow and thymus and 96%
of engrafting cells in female spleen were quiescent (not killed
by hydroxyurea). These data indicate that of the cells from
FU-6 marrow, which engraft in marrow, spleen or thymus,
few are proliferating.
DISCUSSION
Murine hematopoietic pluripotent renewal stem cells have
been defined by their capacity to engraft and repopulate in
an irradiated syngeneic mouse.” Conventionally, onecell
population has been injected at relatively low cell numbers
and blood cell repopulation assessed at different times after
marrow infusion; in some cases two cell populations have
been compared for repopulation in the same irradiatedhost-
Table 1. Engraftment of Marrow From5FU-Treated Mice
Percent Engraftment 2 SEM
Donor
Marrow
No. of
Recipients
Marrow
Spleen
Thymus
Normal
FU-6
FU-35
16-17
19-20
19
29 f 2
7 2 1
34 f 3
23 2 2
521
15 z 1
38 5 5
622
34 f 7
Percentage of male DNA in bone marrow of recipient female mice
given repetitive transplants of normal, FU-6, or FU-35 bone marrow.
Female mice received cells equivalent to two tibiaeand two femurs
for 5 days and were killed at 7 or 10 weeksposttransplantation. Southern blots were probed with pY2 and the percentage engraftment was
calculated using phosphorimage analysis, taking a male control as
100% and a female control as 0%. All samples were corrected for
loading with an IL-3 probe. Statistical analysis showed significant
differences between FU-6 and normal (P< .02) and FU-6 and FU-35
( P< .051 for all three tissues.
Abbreviation: SEM, standard error of the mean.
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ENGRAFTMENT OFCYCLINGMARROWCELLS
927
Bom Marrow
Table 3. Engraftment of Marrow FromDonor Mice Treated With
5FU and PBS or 5FU and Hydroxyurea
Percent Engraftment ? SEM
No
nf
.
"
1
2
3
4
5
6
7
8
9
IO
e
9 9
0
- 0
"""-0
.-
% Engrrmnenc
70
W
117 38
19
44
Y 48 MI 62
Hydroayuraac.td dawm
1
2
3
4
5
6
7
8
9
IO
e
9 9
w
Donor Marrow
Recipients
Marrow
Thymus
Spleen
FU-6 + PBS
FU-6 + hydroxyurea
10
10
4 2 0
375 12 021
721
7 2 2
Percentage of male DNA in bone marrow of recipient female mice
given repetitive transplants of bone marrow from male mice given
5FU on day 0 and theneither PBS or hydroxyurea (900 mgkg) on day
6, 2 hours before being killed. Female mice received cells equivalent
to two tibiae and two femurs for 5 days and were killed at 6 weeks
posttransplantation. Southern blots were probed with pY2 and the
percentage engraftment was calculated using phosphorimage analysis, taking a male control as 100% and a female control as 0%. All
samples were corrected for loading withan IL-3 probe. For all tissues
analyzed, statistical analysis (Wilcoxon's matched pairs test) showed
no significant differences between the two groups.
71 79
Fig 2. Male cells within the bone marrow of noncytoablatedfemale miceafter transplants. Mice were transplanted repetitively for
o
andt w o femurs from donor
5 days with cells equivalent t o t wtibiae
males treated with hydroxyurea (900 mglkg) orsaline (controls) and
analyzed 12 weeks posttransplant. MaleDNA on Southern blots was
detected with the pY2 probe andpercentage engraftment wasquantitated by phosphorimage analysis. Statistical analysis showed no
significant difference between the engraftment from hydroxyureatreated donors and donor mice that hadreceived saline.
a competition assay.".".*' Repopulation of the irradiated host
in the setting of infusion of low numbers of stem cells is.
not surprisingly, ~ l i g o c l o n a l . *The
~ ~ irradiated
~~
transplanted
host represents an amplification system, providing a high
yield from small numbers of cells. It also represents a system
in which there has been cytotoxic damage to the microenvironmental supporting cells of the bone marrow." This is
very different from the model of transplantation into the
normal, unprepared host. This model represents a competi-
Table 2. Engraftment of Marrow From
Hvdroxvurea-Treated Donor Mice
Percent Engraftment 2 SEM
No. of
Donor Marrow
PBS
Hydroxyurea
Recipients
Marrow
Spleen
Thymus
10
10
5327
5718
2522
2325
3717
3325
Percentage of male DNA in bone marrow of recipient female mice
given repetitive transplants of bone marrow from male mice given
either PBS or hydroxyurea (900 mg/kg) 2 hours before being killed.
Female mice received cells equivalent to twotibiae and 2 femurs for
5 days and were killed at 12 weeks posttransplantation. Southern
blots were probed with pY2 and the percentage engraftment was
calculated using phosphorimage analysis, taking a male control as
100% and a female control as 0%. All samples were corrected for
loading with an 11-3 probe. There is no significant difference (Wilcoxon's matched pairs test) between the two groups of samples for
each tissue.
tive situation in whichrelatively large numbers of donor
marrow cells compete with host marrow. Inthis model, there
is no microenvironmental damage and no significant amplification of cells.
Previous studies have indicated that marrow procured 6
days after 5FU competes effectively with normal marrow in
the irradiated transplant,"'."but is defective in theunprepared host.' It has to be noted, however, that these competition assays were performed using C57BU6 mice and thus
may not be comparable to the nonirradiated transplant data
in which studies were performed in the BALBlc strain. This
difference in engraftment could be because the two models
assay intrinsically different stem cells, because stroma is
damaged in the irradiated model or because one system monitors a large readout from a few very proliferative cells. Our
alternative model assesses the results of competition from
relatively large numbers of cells. We are currently comparing
various mouse strains and the effects of 5FU-treated donor
marrow in bothirradiated and nonirradiated hosts. One study
from Harrison et al" reports that post-5FU splenic stem cells
do not compete effectively with normal splenic stem cells
in a long-term repopulation assay in irradiated hosts. These
data could be interpreted as damage to the stem cells by
5FU, but could also be consistent with the present results
indicating a transient functional engraftment defect after
5FU.
The present data show that the 5FU engraftment defect,
which temporally correlates with stem cell proliferation, is
transient and not presentat 35 days after 5FU. This suggests
that the engraftment defect may reflect a relationship between stem cell cycle status and engraftment in this model.
Other work from our laboratory'' has indicated that when
marrow cells are exposed to cytokines, they enter cell cycle
and lose their ability to engraft in unprepared mice. This is
also consistent with a relationship between stem cell cycle
status and engraftment potential. The actual mechanism of
engraftment is still unknown. It could be thatthe engraftment
is determined by antigens such as H-Y or other proteins,
perhaps integrins, which are modulated by 5FU andor cell
cycle status of the cells.
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928
5FU is a chemotherapeutic agent that dramatically reduces
the number of myeloid and lymphoid cells in normal mice.
A major effect of 5FU is to block the enzyme thymidine
synthetase, which, by methylation, converts deoxyuridine
phosphate to deoxythymidine phosphate. When deprived of
deoxythymidine phosphate, cells are unable to synthesize
DNA and accumulate at the beginning of S phase. Doses of
5FU of 30 and 100 mglkg cause an irreversible decrease of
mitotic and 3HUdR labeling indices. No recoveryisseen
over 48 hours, showing that there hasbeen cell death.27
Treatment of mice with 5FU results in a marrow population
relatively enriched for the primitive in vitro progenitor
termed the HPP-CFC.7-9The most primitive marrow stem
cells appear to be dormant and spared by 5FU exposure.
Proliferating hematopoietic cells disappear within 2 daysZX
and, consequently, 5FU treatment induces cycling of primitive engrafting cells. l The engraftment defect seen after 5FU
could, of course, be caused by direct toxic damage to stem
cells or an induced differentiation separate from their cycle
status. The recovery of the defect over time speaks against,
but does not exclude, these possibilities.
If the defect in engraftment seen in marrowcells harvested
from mice after pretreatment with 5FU is related to the proliferative status of these cells, then it would follow that cells
that do engraft in the nonirradiated host are in a quiescent
state. We have assessed the cycle status of these stem cells
using selective killing of cells in S phase by hydroxyurea.
These data indicate that the normal, nonperturbed marrow
stem cells, which engraft in unirradiated hosts, are not proliferating. Similarly, the bulk of FU-6 cells, which engraft, are
not killed by hydroxyurea. These data, in toto, indicate that
actively cycling stem cells do not engraft well in unprepared
hosts.
Using irradiated hosts, others have reported cell cyclerelated alterations in hematopoietic stemcell phenotype.
Early studies on the seeding efficiency of CFU-S in the
spleen showed that a number of in vivo perturbations leading
to stem cell renewal/proliferation including exposure to vinblastine," marrow transplantation3"." or
endotoxin3*led to
reduced seeding efficiency. When fetal was compared with
adu1t33.34 or large compared withsmall
colony-forming
units," the former in each case, shows reduced seeding. One
reason for size differences in stem cells relates to position
in cell
Monette and
DeMello"
addressed the refationship of proliferative status and engraftment directly comparing seeding efficiency in spleen of colony-forming cells
from normal, regenerating and velocity-sedimented cycling
and noncycling marrow preparations. Colony forming cells
in cycle were found to exhibit a 50% reduction in splenic
seeding when compared with normal marrow or sedimented
noncycling cells. We have shown that coincident with endotoxin induced proliferation of CFU-S and granulocytemacrophage colony-forming cell, the transplantation seeding efficiency (f-fraction) of CFU-Swas
decreased by
approximately 30%.4",4'One difficultywith
interpreting
these studies in irradiated hosts is that CFU-S are a heterogeneous population and, at best, only a percentage of these
cells probably represent long-term repopulating cells.
More recently, using a highly purified population (Thy-
RAMSHAW ET AL
1. ll"Lin"'"Sca- 1 +) of hematopoietic stem cells, Fleming et
fractionated Thy-I.l'"Lin"%ca-l+ on the basis of
Hoechst 33342 staining and then compared 100 Go/GIcells
with 100 S/GJM cells for radioprotection and donor derived,
multilineage reconstitution in peripheral blood. A total of
100 Go/GI cells protected 90% of lethally irradiated recipients, whereas the 100 S/G,/M cells protected only 22% of
recipients. In mice 6 months after reconstitution with Go/G,
cells, 34% of B cells, 10% of myeloid cells, and 19% of T
cells were donor. In recipients of S/G,/M cells, only 7.8%
B cells and less than 2.0% of both myeloid and T cells were
donor derived. Thus, the engraftment of hematopoietic stem
cells in the myeloablated host may also be related to cell
cycle status of these cells.
We have previously shown that engraftment of either FU6 or normal marrow in unirradiated
hosts was equally reflected in T cells, B cells, andmyeloid cells, butdid not
. ~ and Keating'
analyze peripheral blood in these s t u d i e ~Wu
have performed studies in untreated recipients indicating that
stem cell engraftment may not correlate with cell differentiation. Our studies usingFISH indicate, in fact, that engraftment seen in marrow, spleen, and thymus for recipients
of normal, FU-6, and FU-35 bone marrow infusions is mirrored by the peripheral blood cells.
Altogether, the present data indicate that the stem cell that
engrafts in the noncytoablated host is a dormant, noncycling
cell and suggest that the impact of 5FU on engraftment may
relate to inducing dormant stem cells into cell cycle. This
type of defect mightbemissedin
the standard irradiated
mouse transplant model where a small number of cells can
repopulate. These data further suggest major phenotypic
changes in stem cells based on their cell cycle status. The
recovery of engraftment after 5FU suggests that the engraftment defect is not due to a toxic effect or differentiation
of the cells. One possibility is that the stem cell differentiation and proliferation phenotype, at least at the more primitive stem cell levels, is dependent on phase of the cell cycle
and not determined in a hierarchial fashion.
a142
ACKNOWLEDGMENT
We thanklab colleagues for helpwithmouse work, especially
Phil Lowry, Donna Deacon, Candi Tiarks, and Maureen Blomberg;
Judy Reilly, Maria Pallavicini (UCSF), and Tom Montoya (UCSF)
for help with the FISH, Tod Turner for maintaining theanima1
facility; and Maria Pallavicini for critical reading of the manuscript.
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1995 86: 924-929
Engraftment of bone marrow cells into normal unprepared hosts:
effects of 5-fluorouracil and cell cycle status
HS Ramshaw, SS Rao, RB Crittenden, SO Peters, HU Weier and PJ Quesenberry
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