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1993 82: 2031-2037
MG Cipolleschi, P Dello Sbarba and M Olivotto The role of hypoxia in the maintenance of hematopoietic stem cells
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The Role of Hypoxia in the Maintenance of Hematopoietic Stem Cells
By Maria Grazia Cipolleschi, Persio
Dello
Sbarba, and Massimo Olivotto
Bone marrow cell liquid cu ltures were incubated at various
oxygen concentrations ranging from
0
o 18 (air). The
total number of cells in cu lture (CT) at the end of a 6-day
incubationwas found to be directly proportional to the oxy-
gen concentration.
A s
compared wit h air-incubated con-
trols, cells recovered from severely hypoxic (1 oxygen)
day-5 liquid cu ltures showed (1) the same day-7 colony-
formation efficiency in semisolid culture (neutrophilic/
monocytic colonies) or in spleen; (2)a higher day-14 spleen
colony -formation efficiency; (3)an enhanced radio-protec-
tion ability; and (4)an increased marrow repopulation abil-
ity, as measured by determining either total cell number in
recipient marrow
MRk, , ,
or the capacity of the latter of
generating day-7 neutrophi lic/monocytic colonies in sec-
LTHOUGH developmental and cytokinetic properties
A
of hematopoietic stem
cells
have been intensely stud-
ied, the in vivo organization of the hematopoietic tissue
remains largely unknown. In particular, the mechanisms
regulating the life-long maintenance of stem cells are still
unexplained. According to a widely accepted view, the con-
ditions for this maintenance are realized in physiologically
segregated areas of bone marrow (BM) (“niches”), wherein
stem cells are restrained from commitment to extensive pro-
liferation and differentiation.
In a previous report, we proposed that these niches are
hypoxic areas, in which only oxygen-independent cells are
able to survive.* This proposal was based on the following
rationale. Arterial blood enters the highly branched network
of medullary sinuses only after circulating in the cortical
canalicular sy~tem,~
o
that a relatively desaturated blood
reaches the marrow. Indeed, average oxygen tension in BM
blood is significantly ower than in other organs and tissues,
being similar to that in the jugular vein Moreover, it is
likely that cells crowding around sinuses strongly compete
for the already scarce oxygen supplied, resulting in an even
lower oxygen tension within the core of cell mass.5 It is
highly plausible that in such areas the proliferation of stem
cells is blocked without threatening their survival. In fact,
while mitochondrial respiration seems a prerequisite for
cells to enter the mitotic ~ y c l e , ~ - ~rowth factors have been
shown to sustain cell survival through the stimulation of
glycoly~is.~-~’herefore, the deeply hypoxic areas of BM
appear to be particularly suitable for the long-term mainte-
nance of stem cells, whereas the better oxygenated areas
would allow proliferation of more differentiated progeni-
tors.
In keeping with our hypothesis, hematopoietic progeni-
tors actually appear to be aligned along the oxygen gradient
of the BM. Progenitors responsible for BM repopulation
have in fact been shown to be more concentrated in the low
oxygen areas of BM,” whereas fast cycling neutrophilic/
monocytic colony-formingunits (CFU-NM) and day-7 col-
ony-forming units in spleen (CFU-S,) reside preferentially
in subendosteal areas, where the blood enters the BM circu-
lation. Furthermore, the probability of progenitors to be
recruited into the mitotic cycle seems inversely related to
the distance from those areas.l3-I5
ondary in vitro assays (MRA,,u.,M). Taking into account
CT, the absolute numbers of progenitors in cu lture were
also computed. The results showed that, w it h respect to
time
0,
incubation n air produced an increase in the num-
ber of day-7 CFUs and a decrease in the number of the
other progenitors , whereas in hypoxic cultures all types of
progenitorsdecreased. However, as compared wi th air-in-
cubated controls, all progenitors, except cells sustaining
MR FU.NM,
were reduced in hypoxic cultures. The degree
of reduction paralleled the position of t he progenitor n the
hematopoietic hierarchy, being maximum for day-7 CFUs
and null for cells sustaining
MRACFU.NM,
which, in fact,
were better preserved in hypoxic cultures.
0 1993by The American Society of Hematology
In our laboratory, the respiration-dependence of BM cells
(BMC) was indirectly studied in vitro by treating short-term
liquid cultures with an excess of pyruvate or other oxidiz-
able substrates. This excess, saturating the respiratory chain,
produces the impairment of oxidative limiting steps of
various metabolic pathways, thus mimicking the effects of
inhibitors of mitochondrial respiration.16 The addition of
pyruvate reduces colony formation from CFU-NM, with-
out affecting their in vitro generation from stem cells, sug-
gesting differences between CFU-NM and their progenitors
in the dependence on mitochondrial respiration.2
In this report, we present a study of the effects of reduced
oxygen tension on mass liquid cultures of mouse BMC. We
provide evidence that oxygen tension is a critical factor for
BMC expansion in vitro and that the different types of he-
matopoietic progenitors display a varying degree of sensitiv-
ity to hypoxia. The lower this sensitivity, the higher the hier-
archical level of the progenitor,
so
that incubation in severe
hypoxia leads to a substantial concentration of some types
of progenitors within the population and selectively pre-
serves progenitors of the highest hierarchical level.
MATERIALS AND METHODS
Cell recovery and preparation.
BMC
were obtained from
pooled 8- to 12-week-old
CBA T6T6
mice by forced injection of
RPMI-I640 medium
(GIBCO
Ltd, Paisley,
UK)
into femoral bone
From the Istituto di P atologia Generale, Universitd di Firenze,
Submitted October 6 , 1992; accepted June 2, 1993.
Supported b y grants from Associazione Italiana per la Ricerca
SU
Cancro (AIR C), Consiglio Nazionale delle Ricerche (Project “Appli-
cazioni Cliniche della Ricerca Oncologica’), and Minister0 della
Universitd e della Ricerca Scientifica e Tecnologica (M UR ST , ondi
60 e 40%).
Address reprint requests to Massimo Olivotto, MD Istituto di
Patologia Generale viale G.B. Morgagni 50, I-50134 Firenze, Italy.
Th e publication costs of this article were defrayed in part by page
charge paymen t. This article must therefore be hereby marked
“advertisement” n accordanc e with 18 U.S.C.section I734 solely to
indicate this fact.
Florence, Italy.
993 by T he American Society ofHematology.
0006-49 71/93/8207-00I0 3.00/0
Blood,
Vol82, No 7 (October 1 , 1993: pp 2031 2037
203 1
8/17/2019 Blood 1993 Cipolleschi 2031 7
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2 32
CIPOLLESCHI, DELL0 SBARBA, AND OLIVOTTO
shafts. Cells were then centrifuged (250g for 10 minutes), resus-
pended in 0.87% NH4CI in H 2 0 o lyse erythrocytes, washed, and
plated in cell culture dishes in RPMI-1640 supplemented with 10%
heat-inactivated horse serum (HS; Flow Laboratories, Irvine,
UK).
After 3 hours of incubation, nonadherent cells were recovered,
counted, and transferred into liquid or semisolid cultures (see be-
low). Cell counts were performed in a hemocytometer and cell via-
bility estimated by the trypan blue exclusion test, diluting cell sus-
pensions
I :
with a 0.4% wt/vol trypan solution in
0.85
saline
(Flow Laboratories).
Spleen conditioned medi um
SCM). Mouse SCM was prepared
and tested for BMC growth-stimulatory activity as described else-
where.’ In brief, 10’ mouse splenocytes were cultured in 50 mL of
RPMI- 1640 supplemented with 5 pooled, heat-inactivated hu-
man serum and 0.4% pokeweed mitogen solution (GIBCO); after
7
days of incubation, culture medium was recovered, filtered, and
frozen in small aliquots to be used without further manipulation.
BMC culture in liquid medium was per-
formed in gas leak-proof culture vessels that allowed us to establish,
and maintain throughout the incubation, atmospheres composed
of 5% C02and different proportions of oxygen (ranging from0% o
10%)and nitrogen
(95
to
85 ).”*18
BMC were plated in 250-mL
glass flasks, in RPMI-1640 supplemented with 20% HS and 10%
SCM ( 5 X IO6 cells in 50 mL per flask). After flushing he sterile-fil-
tered gas mixture, the air-tight inlet and outlet were closed and the
flasks transferred into an incubator at 37°C. Control cultures were
established n the same type
of
flask, but kept in direct communica-
tion with the atmosphere of the incubator: 95 air, 5% C02,water-
saturated (incubation “in air”). The pH of the hypoxic cultures
remained within physiologic limits (7.2 to 7.4) hroughout the incu-
bation.
After
5
to 6 days of incubation in hypoxia or in air, cells were
washed, counted, and diluted appropriately for the in vitro or in
vivo assays, as indicated below. BMC recovered directly from ani-
mals (“time-zero” of the cultures) were assayed under the same
conditions in a larger set of separate experiments.
In
vitro clonal assay.
To estimate the number of CFU-NM in
BMC populations, semisolid cultures were established by plating
IO4
viable cells per 3-cm petri dish in mL of RPMI-I640 supple-
mented with 20%
HS,
10% SCM, and 0.3% (wt/vol) agar (Bacto
Agar; DIFCO, Detroit, MI). Assays were performed in triplicate
and always incubated in air. After 7 days ofincubation, the number
of colonies (aggregates of 50 or more cells)per dish was scored at 25
X magnification. Colony-formationefficiency (CFE) was expressed
as the ratio of the number of colonies developed to the number of
cells plated per dish.
Cell culture sy stem.
In Vivo Assays
Cells were transplanted into pooled
8-
to 12-week-old, syngeneic
mice that had been lethally irradiated with a total dose of 10
C y
at a
rate of
1
Gy/min from a 6oCo, 1.2 MeV source (Theratron 780;
Atomic Energy of Canada Ltd, Ottawa, Canada). Cell suspensions
in serum-free culture medium were injected into the lateral tail vein
within 3 hours after irradiation. Irradiated mice injected with cul-
ture medium only were used to determine the background level for
each parameter studied.
Cells were transplanted into 3
to 5 mice per group and spleens recovered from recipient mice
7
(CFU-S,) or 14 days (CFU-SI.,) after transplantation. Spleens were
then fixed in Bouin’s solution, and macroscopic surface colonies
counted at
2 X
magnification. To determine the number of CFU-
S I 4 ,
chosen as an assay for the “delayed” spleen colony-forming
activity,lS2’various cell suspensionswere transplanted(2.5 X lo4 o
Spleen colony-formation assays.
5 x IO cells/0.2 mL). Counts of large day-14 spleen colonies, a
possible source of inacc~racy ,~~ere confirmed by determining
spleen weight (not shown); CFE was expressed as the ratio of the
colony number to the number of cells transplanted.
The radio-
protective ability (RPA) of BMC populations, ie, the capacity to
confer long-term survival to lethally irradiated mice, was measured
by transplanting various cell suspensions (2 X
IO4
to 2
X
IO6 cells/
0.2 mL), usually into I O mice per suspension.The fraction ofrecipi-
ent mice surviving 30 days after transplantation was determined for
each suspension. RPA was expressed as the ratio of the percentage
of surviving mice to the number of transplanted cells.z”6
BM repopulating ability ( M U ) of BMC populations was mea-
sured basically as described by Hodgson et al;,,’’ by transplanting
various cell suspensions
5 x
io4 o 2x IO6 cellsj0.2 mL) into 3 to
5
mice per suspension. Femurs were recovered from recipient mice
14 days after transplantation and individually processed. The total
number of nucleated cells per femur was determined and aliquots
of cells were assayed for the presence of CFU-NM, as described
above. MRA was expressed as the ratio of the total number of cells
(MRAeII), r the number of CFU-NM (MRA,-m-NM), per recipient
femur, to the number oftransplanted cells. MRA was chosen
as an assay for MRA,-,
.20,28s29
Data obtained with increasing dose of transplanted cells were
plotted in log/iog scale and best fitted by linear regression, with the
exclusion of plateau values, according to Hodgson et a1.28
Cells responsible for RPA
or
MRAel,,m-NM were referred to as
Radioprotection and marrow repopulation assays.
RPA cells or MRAcell,CFU.NM
e[ls.20.25.26.29
RESULTS
Dependence
of BMC Growth on
Oxygen Tension
Total number
of
cells (CT)was determined in BMC liq-
uid cultures incubated
for
6 days at different oxygen ten-
sions. CT resulted directly proportional to oxygen concen-
tration
of
the incubation atmosphere, increasing, as
compared with the time-0 value, within an oxygen concen-
tration range
of
3 o 18 (Fig 1). There was no increase in
cell number at 3 oxygen, whereas cell loss occurred at
lower concentrations.
0
5
10
15
20
percentage
of
oxygen
Fig 1.
Effects of oxygen tension on BMC grow th. CT in BMC
liquid cultures was determined at t he end of 6 days of incubation.
Points are means standard errors of 11 separate experiments.
Dashed line indicates CT value at ti me 0.
8/17/2019 Blood 1993 Cipolleschi 2031 7
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HYPOXIA PRESERVES HEMATOPOIETIC STEM CELLS
2033
The data indicated that, whereas BMC expansion in vitro
is oxygen-dependent, a sizeable number of hematopoietic
cells is able to survive in severe hypoxia (1 to
3 ).
We
decided to characterize he composition ofthis residual pop-
ulation with respect to several types of hematopoietic pro-
genitors and stem cells. Incubation in 1 oxygen for
5
days,
providing sufficient numbers of viable cells for secondary in
vitro or in vivo assays, was chosen on the basis of prelimi-
nary tests (not shown) and used for all experiments reported
below.
Effects of Hypoxia on BMC Cloning Eficiency
Table 1 shows the effect of a 5-day incubation of BMC in
1 oxygen (hypoxia) or in air (aerobiosis) on cloning effi-
ciency, as detected 7 days after replating in vitro (CFU-NM)
or transplantation in vivo (CFU-S7).The in vitro clonal as-
says were performed in both cases in air, to avoid differences
in yield caused by the reported influence of oxygen tension
on colony formation in semisolid Day-7 clon-
ing efficiency, both in vitro and in spleen, was found to be
significantly decreased,
as
compared with time-zero, inde-
pendently of the incubation atmosphere. Furthermore, no
significant effect on cloning efficiency was produced by in-
cubation in
1
oxygen as compared with air. This indicated
that hypoxia, although reducing CT (Fig
l) ,
did not alter the
concentrations of CFU-NM and CFU-S,, and, therefore,
that these progenitors are as sensitive to hypoxia as the over-
all BMC population.
On the contrary, BMC cloning efficiency in spleen, as
detected 14 days after transplantation (CFU-S14),was signifi-
cantly influenced by incubation in hypoxia. This is shown
in Fig
2,
in which the relationship between inoculum size
and colony number is reported for all tested conditions.
This relationship turned out to be linear in all cases, allow-
ing for the calculation of the cloning efficiencies from the
slopes of the lines. Cloning efficiency was found to be de-
creased in day-5 cultures, as compared with time 0, indepen-
dently of the incubation atmosphere. However, this reduc-
tion was
5
times lower in hypoxia than in aerobiosis. Thus,
Table 1. Effects of BMC Incubation in Air or Hypoxia on Day-7
Cloning Efficiency in Culture (CFU-NM) or in Spleen (CFU-S)
Incubation
ColoniesPer
Dish Colonies Per Spleen
No time 0)
In air
In
1
oxygen
34.9? 1.63(39; 0)
21.2 ? 5.39 26; 7)
22 3
k
1.94
(1
9; )
15.2 0 75 (56; 11)
4.5 ? 0.80t (27; 6)
6.8 ? 0.89t
(1
7; 3)
BMC, recovered directly from donor mice (time 0) or from liquidCUI-
tures incubated or 5 days in either air (control) or hypoxia
(1
oxygen),
were replated in semisolid medium
(1O4
cells per dish) or transplanted
into lethally irradiated syngeneic mice (1O cells per mouse), and colo-
nies counted 7 days later. Data are means ? intraexperiment standard
errors (total number of counts; number of separate experiments). Re-
sults were assessed by one-way analysis of variance. Differences be-
tween time 0 and day-5 cultures are defined by the symbols. Differences
between hypoxic and control day-5 cultures were not significant.
P
<
.01 (71
degrees of freedom).
t P
< .01 88 degrees of freedom).
8
6
2
c n 4
LL
0 2
0
r
1
2
3 4
5
cel ls t ransplanted ( X ~ O - ~ )
Fig 2.
Dose-response relationship between the number of BMC
transplanted and the number
of
day-14 spleen colonies (CFU-S,J.
Cells to be transplanted were recovered directly from donor mice
(time0; A) or from 5-day cultures incubated in hypoxia (1 oxygen;
0 or in air 0 ) . he main comparison (air
w
hypoxia) was balanced.
Points are means
?
standard errors of counts from three separate
and independent experiments. Lines wer e We d by linear regres-
sion on all experimental counts (time zero, 28; hypoxia,32;air, 24) .
The slopes of th e lines, corresponding approximately to co lony-for-
mation efficiencies, were 13.2 8 ? 4.18; 4.90 -C 0.66; 0.97 -C
0.1
6,
respectively. Differences between slopes were significant
according to th e Student's t-test (time0 wair P
<
.001,47 degrees
of freedom; tim e
0
w
hypoxiaP
<
.05 ,51 degrees of freedom; air
w
hypoxia
P
< .0 01 ,4 6 degrees of freedom).
CFU-SI4appeared concentrated in hypoxic as compared
with aerobic day-5 cultures, showing a substantial differ-
ence between these progenitors and CFU-NM or
CFU-s,.
Effects
of
Hypoxia on
RPA
The percent survival of lethally irradiated mice is plotted
in Fig 3 versus the inoculum size. To compare variously
treated cell populations, we considered intercepts of the
lines best fitting the data with the number of transplanted
cells required to obtain 50% survival. This number was esti-
mated to correspond approximately to
3.5 X
lo cells at
time zero,
I 2
X
O4 cells incubated in hypoxia, and
69
X
O4
cells incubated in air. Thus, RPA was found decreased in
day-5 cultures, as compared with time
0,
but this decrease
was 5.7 times lower in hypoxia than in aerobiosis, parallel-
ing the results obtained for CFU-Sl4.
Effects
of
Hypoxia
on
MRA
MRA,,, was calculated by plotting the number of cells
recovered from recipient femoral marrow versus the num-
ber of transplanted cells (Fig 4A). To compare variously
treated cell populations, we considered intercepts of the
lines fitting the data with a reference value of repopulation
of recipient femoral marrow (2.5 X lo5cells, corresponding
to the dashed line in Fig 4A). It was estimated that, to obtain
this value, one needs 8.7
X
lo cells at time
0,
18.2 X
lo4
8/17/2019 Blood 1993 Cipolleschi 2031 7
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2034
6 0
L
2
5 5
a
e
-
5 0
2
UI
-
4.5
CIPOLLESCHL DELL0 SBARBA, AND OLIVOTTO
2 0
-
a
f
a
>
.-
I)
1.5.
e
log cells transplanted
Fig 3. Dose-response relationship between the number of BMC
transplanted and the percentage of recipient mice surviving 30
days after transplantation (RPA). Cells to be transplanted were re-
covered directly f rom donor mice (time 0;
A)
r from 5-day cultures
incubated n hypoxia (1 oxygen;
0)
or in air
0 ) .
he main compari-
son (air
v
hypoxia) was balanced. Points represent percent survival,
calculated on groups o f 10 mice per point. Lines were fitted by
linear regression, wit h the exclusion of plateau values. Dashed ine
indicates 50% survival.
cells incubated in hypoxia, an d 138 X lo cells incubated in
air. Thus, again, MRA,, decreased in culture, but this de-
crease was 7.6 times lower in hypoxia th an in aerobiosis, in
keeping with the results obtained for
CFU-SI,
and RPA.
T h e a b o v e p r o c e d u r e w a s a p p l i e d t o e s t i m a t e
MRA,-,-,,
(Fig 4B), assuming IO3 CFU-NM as a reference
value of repopulation of recipient femoral marrow (dashed
line in Fig 4B). Thi s value was obtained with 14.8 X IO4 cells
at tim e zero, 9.5
X
lo4cells incubated in hypoxia, an d 120 X
IO4 cells incubated in air. Strikingly, in hypoxic cultures,
MRA,,,,
not only was much higher than i n aerobic cul-
tures (12.6 times), but it also appeared unreduced, if not
enhanced, in comparison to time 0.
Efects o Hypoxia on the Absolute Number of Progenitors
n Culture
Th e effects of hypoxia
on
the absolute numbers of hema-
topoietic progenitors were tentatively quantified by taking
into account th e changes in C T under the various culture
conditions (Table 2). It was shown tha t incubation in air for
5 days resulted in an increase in the num ber of CFU-NM
and CFU-S,, along with
CT
expansion. O n the other hand,
all the other progenitors decreased: CFU-SI,,
RPA
cells,
and MRA,,, cells (prog enitors sustaining RPA and
M W , , ,
respectively) to 26 . on the average, whereas
MRACN-NM
cells (progenitors responsible for MRACN-NM) to about
50 . Incubation in hypoxia for 5 days reduced all types of
progenitors to 2 I , on t he average, the only exception being
MRA,,,
cells, that were abo ut 75 conserved.
Comparison of columns 2 an d 3 (or 4 and 5) of Table 2
led to the conclusion that the enrichment of hypoxic cul-
tures with hematopoietic progenitors shown in Figs 2, 3,
1
A
A
4 01
4
6
6 7
log,, cells transplanted
4 0
r
1.5
1
4 5
6
7
log,, cells transplanted
Fig 4. Dose-response relationship between the number of BMC
transplanted and the number of nucleated cells (A) or CFU-NM (B)
recovered from each femur of recipient animals. Cells to be trans-
planted were recovered directly from donor mice (time0;
A
r from
5-day cultures incubated n hypoxia 1%oxygen;0)or inair
0 ) .
he
main comparison (air v hypoxia)was balanced. Points are means -C
standard error of two to five separate and independent experi-
ments. Lines were fitted by linear regression, wi th the exclusion of
plateau values. Dashed lines indicate repopulation of recipient fe-
mur with 2.5 X
1O
nucleated cells (A) or
1O3
CFU-NM (B). One-
way analysis of variance was performed on data normalized or a 5
x
1
O5
cell inoculum as follows:
5 x 106
loglo Cells Transplanted
where N is the number of cells (A)or CFU-NM (B) per femur. This
normalization gave (mean f SEM, total number of experimental
points, number of separate experiments) (A) time 0,3.19 f 0.07,
44, 8; hypoxia, 2.76 0.09,28, 7; air, 1.62 f 0.1 1, 29, 6; (B)
time0,3.44
-C
0.08.35.6; hypoxia,3.68 0.15,21,5;air,2.44
f 0.16, 12, 4. Differences between the above means were
(A)
time
0
versus air,
<
.001; time 0 versus hypoxia, <
.01;
air
versus hypoxia, P <
.001;
(B) time 0 versus air,
C
,001; ime 0
versus hypoxia, not significant; air versus hypoxia, P < .001.
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HYPOXIA PRESERVES HEMATOPOIETIC STEM CELLS 2035
Table 2. Computation of Total Number of Hematopoietic Progenitors in BMC Cultures
Absolute
Number of Cells in Culture o f t = 0 Values
Type of
Progenitor
t = O Air Hypoxia Air Hypoxia
~
Culture no.
Cells (CT)
CFU-NM’
CFU-S,
CFU-SI,’
RPA cellst§
MRA cells$§
MRA cells*§
_ _ _ _ ~
1
5
x
lo6
17,450
760
664
143
57
34
2
21.2
x
1 0 6
44,944
954
206
31
15
18
3
2.4
X
lo6
5,352
163
118
20
13
25
_____
4
424.0
257.6
125.5
31.O
21.7
26.3
52.9
___
5
48.0
30.7
21.5
17.8
14.0
22.8
73.5
Abbreviations:t
= 0
(time0 .MC recovered directly from donor mice; air, 5-day cultures incubated n air; hypoxia, 5-day cultures incubated n 1
Values were obtained multiplying CT by the colony-formation efficiencies (see Table and the legend to Fig 2).
t Values are expressed n arbitrary units, 1 U corresponding o the number of transplanted cells supporting survival of 50 ecipient mice after 30
$
Values are expressed in arbitrary units,
1
U corresponding o the number of cells to be transplanted o repopulate each femur of the recipient with
§ Values were obtained dividing CT by the number of transplanted cells corresponding o
1 U,
calculated from Fig3 (RPA), Fig
4A
(MRA,,,,), or Fig48
oxygen.
days.
2.5
x lo5
BMC (MRA,,,,) or
lo3
CFU-NM (MRACFU.NM).
(MRAcFu-NM).
and 4A was essentially accounted for by the suppression in
these cultures of the CT increase that occurs in air.
MRACW-NM cells (Fig 4B) had the further advantage of be-
ing better preserved in hypoxic cultures, rather than in aero-
bic cultures.
DISCUSSION
The main information to emerge from this study was that
hypoxic culture conditions clearly selected for certain types
of hematopoietic progenitors in BMC cultures, as com-
pared with aerobic cultures. This effect was maximal for
MRACmsNMells (Fig 4B). A clearer outline of the fate of
various progenitors n aerobiosisor in hypoxia was obtained
from the computation of their total number in culture (Ta-
ble
2),
showing that
1 )
the concentration
of
most progeni-
tors in hypoxic cultures was essentially accounted for by
suppression of CT increase; and (2) MRApm-NM cells were
better preserved in hypoxia than in aerobiosis.
When total numbers of progenitors in hypoxic cultures
(Table 2) were reported as percentages of the corresponding
number in aerobic cultures (Fig 5 ) , it was possible to iden-
tify three main categories of progenitors:
(1)
oxygen-depen-
dent progenitors, including CFU-NM and CFU-S,; ( 2 )oxy-
gen-independent progenitors (CFU-Sl4, RPA cells, and
MR&,, cells); and
(3)
hypoxia-preserved progenitors
(MRAcW-NM cells).
The above categories fit very well with progenitor pheno-
types identified on the basis of mitochondrial activity, as
measured by rhodamine-
123
(Rh)
(1)
Rh-posi-
tive cells (CFU-S, and persistent-CFU-S,,); (2) Rh-weakly
positive cells (delayed-CFU-S,,, RPA cells, and MRA,,
cells); and
(3)
Rh-dull cells (most MRApFU-NMe11s).19-21~24~29
The finding that CFU-NM and CFU-S, depend on oxy-
gen for survival and expansion in vitro is in keeping with the
abundance of active mitochondria shown in group (1) cells
by the Rh-positivity. This result is also consistent with the
respiration-dependent step in the mitotic cycle shown for
CFU-NM2 and suggests the existence of a similar step in
CFU-S,, that, like CFU-NM, are actively cycling commit-
ted p r o g e n i t ~ r s . ~ ~ , ~ ~ , ~ ~n the other hand, the oxygen-depen-
dence of CFU-NM contrasts with the reported enhance-
ment of colony-formation efficiency in semisolid cultures
incubated in hypoxia, an effect attributed to a reduced oxy-
gen toxicity on clonogenic progenitors.3G34 owever, sensi-
tivity of these cells to oxygen toxicity is apparently lower in
mass liquid cultures than in clonal assays in semisolid me-
d i ~ m . ~ ’ , ~ ’hus, it is conceivable hat in our system the posi-
tive effects of oxygen on cell growth largely prevail over a
low degree of oxygen toxicity.
The low sensitivity to hypoxia of CFU-SI4, RPA cells,
and MRA,” cells (Fig
5)
is in keeping with the weak Rh-pos-
itivity of group
(2)
cells, indicating a scarcity of active mito-
chondria. It is worthwhile to recall that these progenitors are
mainly q~i esc ent ,~,ut readily recruitable and committable
by growth factors (“activated stem cells”) to generate higher
n
cells cells cells
Fig
5.
Effects
of
hypoxia on the absolute number of hematopoi-
etic progenitors in BMC cultures. Bars represent values referring to
hypoxic cultures (reported in column 3 of Table 2 . xpressed as
percentagesof those
of
control cultures in air (column 2 of Table 2).
The latter are indicated by the dashed line (see ext).
8/17/2019 Blood 1993 Cipolleschi 2031 7
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2036
CIPOLLESCHI, DELL0 SBARBA, AND
OLIVOTTO
n umb er s o f le ss immatu r e p r ~g en i t o r s . ~ ~ .~ ’herefore, acti-
vated stem cells are likely to be depleted in o ur system as in
any growth factor-stimulated culture. Indeed, in o ur experi-
ments, these cells were found substantially decreased in
both control and hypoxic cultures, pointing out the irrele-
vance of cell respiration for their survival.
Finally, within the lim its that MW,, , cells (group
3)
are actually Rh-dull, ie, lacking of active mitochondria,
these progenitors could be regarded as anaerobically
adapted. In keeping with this view, MRA cells have been
found to be more highly concentrated in the low oxygen
areas of BM.” Anaerobic adaptation of metabolism, usually
accompanied by an enhanced cell sensitivity to oxygen
toxicity:’ se em s to be confirmed by our finding that
MRA, ce lls w ere be tt er m ain ta in ed in h yp ox ia th an i n
air,
a
striking difference from all of the o ther types of pro-
genitors. Interestingly, MRA, cells are also mostly
n o n c y ~ l i n g , ~ ~ ~ ~ ~ ~ ~ ~n essential attribute of the most imma-
ture hematopoietic progenitors, the quiescent stem ~ells.4~
Th e latter are believed to be less easily depleted than acti-
vated stem cells unde r the action of growth factors, as con-
firmed by ou r data (see Table 2, column 4).
In this context, one can hypothesize that ana erobic adap-
tation of metabolism is a major feature distinguishing
activated from quiescent stem cells. Assuming that the re-
cruitment of stem cells to active hematopoiesis is respira-
tion-dependent, th e lack of active mitochondria in som e of
these cells would prevent their recruitment and limit the
action ofgrowth factors to the support of survival.43 n other
words, the anaerobic orientation of a subset of stem cells
would reduce their sensitivity to proliferation/differentia-
tion stimuli and play a crucial role in their maintenance in
Go
nd long-term conservation.
On the w hole, data presented seem to support the pro-
posed hypothesis that quiescent stem cells survive in “hyp-
oxic niches”
of
hematopoietic tissue, protected from com-
mitment. An increase of number/activity of mitochondria
may well be a prerequisite for the activation of stem cells.
These activated stem cells could conceivably move t o less
hypoxic areas close to the niches, where the response to
growth factors is no longer restrained a nd th e generation of
lineage-restricted progenitors is possible. The latter, in bet-
ter oxygenated areas, would then undergo the extensive pro-
liferation responsible for clonal expansion.
Whatever the merit o fthe se considerations, it is a fact that
various subsets of hematop oietic progenitors are differently
affected by oxygen tension. While this effect needs to be
further explored by BMC fractionation procedures, incuba-
tion
in severe hypoxia or anoxia might prove to be a useful
and “physiologic” tool to select quiescent stem cells from
normal a nd leukemic BM.
ACKNOWLEDGMENT
The authors thank
A.
Fonnesu, MD, chairman of the Institute of
General Pathology, Florence, for help and advice. Thanks are also
due to L. Calorini, MD, Institute of General Pathology, Florence,
for technical assistance; A. Becciolini and the Staff oft he Radiother-
apy Unit, USL 10D, Florence, for advice and assistance to the irra-
diation of mice; V. Boddi, Institute ofG eneral Pathology, Florence,
and P. Urbano, MD , Institute of Microbiology, Florence, for statis-
tical analysis of data; P.A. Bemabei, MD , Dep artmen t of Hema tol-
ogy, USL IOD, Florence,
for
his helpful discussions and critical
reading of the man uscript; and
I.S.
Hawkins, MD, for her revision
of
the English.
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