blood 1993 cipolleschi 2031 7

8/17/2019 Blood 1993 Cipolleschi 2031 7

1/8

1993 82: 2031-2037

MG Cipolleschi, P Dello Sbarba and M Olivotto The role of hypoxia in the maintenance of hematopoietic stem cells

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints

Information about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

reserved.Copyright 2011 by The American Society of Hematology; all rights900, Washington DC 20036.weekly by the American Society of Hematology, 2021 L St, NW, SuiteBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published

For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom For personal use only.by guest on October 8, 2013. bloodjournal.hematologylibrary.orgFrom

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests


2/8

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


3/8

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.


4/8

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


5/8

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.


6/8

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).


7/8

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.

REFERENCES

1. Schofield R: The relationship between the spleen colony-

form ing cell and the haemop oietic stem cell-A hypoth esis. Blood

Cells 4:7, 1 978

2. Dello Sbarba P, Cipolleschi MG , Olivotto M: Hemopoietic

progenitor cells are sen sitive o the cy tostatic effect of pyruvate. Exp

Hem atol 15: 137, 1987

3. Lichtman MA: The ultrastructure ofthe hemopoietic environ-

ment of the marrow: A review. Exp Hematol9 :391, 1981

4. Grant WC, Root WS: The relation of

O

in bone marrow

blood to post-hemorrhagic hemopoiesis. Am J Physiol 150:6

18,

1947

5 Tannock IF: The relation between cell proliferation and the

vascular system in a transplanted mouse mammary tumour. Br J

Cancer 22:258, 1968

6. Ha ms JW, Meyskens F, Patt HM: Biochemical studies of cy-

tokinetic changes during tum or growth. Cancer Res 30:1937, 1970

7. Olivotto

M,

Paoletti F The role of respiration in tumor cell

transition from the non-cycling to the cycling state. J Cell Physiol

107:243, I98 1

8. Olivotto M, Arcangeli A, Caldini R, Chevanne M , Cipolleschi

MG, Dello Sbarba P: Metabolic aspects of cell cycle regulation in

normal and cance r cells. Toxicol Pathol 12:369, 1984

9. Whetton AD, Dexter TM: Effect of haematopoietic cell

growth factor on intracellular ATP levels. Nature 303:629, 1983

10. Whetton AD, Bad1 GW, Dexter TM: Haemopoietic cell

growth factor m ediates cell survival via its action o n glucose trans-

port. EMBO J 3:409, 1984

11.

Dexter TM, Whetton AD, Bazill G W Haemopoietic cell

growth factor and glucose transport: Its role in cell survival and the

relevance

of

this in normal haem opoiesis and leukaemia. Differen-

tiation 27:163, 1984

12. Lord BI: The architecture of bone m arrow cell populations,

in Murphy MJ J r (ed): Concise Reviews in Clinical and Experimen-

tal Hematology. Dayton, OH, AlphaMed, 1992, p 225

13. Lord BI, Testa NG , Hendry J H: Th e relative spatial distribu-

tions of CFU-S and CFU-C in the normal mouse femur. Blood

46:65, 1974

14. Shackney SE, Ford

SS,

Wittig AB: Kmetic-microarchitec-

tural correlations in the bone marrow of the mouse. Cell Tissue

net 8:505, 1975

15.

Mason TM, Lord BI: Hendry JH: T he development of spa-

tial distributions ofCFU -S and

in-vitro

CFU-C in fem ora of mice of

different ages. Br J Haematol 73:455, 1989

16. Olivotto M, Caldini

R,

Chevanne M , Cipolleschi MG: T he

respiration-linked limiting step of tumor cell transition from the

non-cycling to the cycling state: Its inhibition by oxidizable sub-

strates and its relationships to purine metabolism. J Cell Physiol

116:149, 1983

17. Allalunis-Turner MJ, Koch CJ, Chapman JD: Growth of

murine bone marrow under various oxygen conditions in media

buffered with HEPES. Int J Cell Cloning 5:315, 1987

18. Taylor WG, Camalier RF, Sanford

KK:

Density-dependent

effects of oxygen on the growth of mam malian fibroblasts in cul-

ture. J Cell Physiol 95:33, 1978

19. Visser WM, de Vries

P

Isolation of spleen-colony forming


8/8

HYPOXIA PRESERVES HEMATOPOIETIC STEM CELLS

2037

cells (CFU-s) using wheat germ agglutinin and rhodamine 123 abel-

ing. Blood Cells 14:369, 1988

20. Ploemacher RE, Brons NHC Cells with marrow and spleen

repopulating ability and forming spleen colonies on day 16, 12, and

8 are sequentially ordered on the basis ofincreasing rhodamine 123

retention. J Cell Physiol 136531, 1988

2 1. Ploemacher RE, Brons NHC In vivo proliferative and dif-

ferential properties of murine bone marrow cells separated on the

basis of rhodamine 123 retention. Exp Hematol 16:903, 1988

22. van Zant G Studies of hematopoietic stem cells spared by

5-fluorouracil. J Exp Med 159:679, 1984

23. Necas E, Znojil V: A comparison of stem cell assays using

early or late spleen colonies. Cell Tissue net 22: 1

1 I ,

1989

24. Mulder AH, Visser JWM: Separation and functional analy-

sis of bone marrow cells separated by rhodamine-123 fluorescence.

Exp Hematol 15:99, 1987

25. Ploemacher RE, Brons NHC: Isolation of hemopoietic stem

cell subsets from murine bone marrow: I. Radioprotectiveability of

purified cell suspensions differing in the proportion of day-7 and

day-12 CFU-S. Exp Hematol 16:21, 1988

26. Ploemacher RE, Brons NHC Isolation of hemopoietic stem

cell subsets from murine bone marrow: 11. Evidence for an early

precursor of day-12 CFU-S and cells associated with radioprotec-

tive ability. Exp Hematol 16:27, 1988

27. Hodgson GS, Bradley TR: Properties of haematopoietic

stem cells surviving 5-fluorouracil treatment: Evidence for a pre-

CFU-S cell? Nature 281:381, 1979

28. Hodgson GS, Bradley TR, Radley JM: The organization of

hemopoietic tissue as inferred from the effects of 5-fluorouracil.

Exp Hematol 10:26, 1982

29. Ploemacher RE, Brons NHC: Separation

of

CFU-S from

primitive cells responsible for reconstitution of the bone marrow

hemopoietic stem cell compartment following irradiation: Evi-

dence for a pre-CFU-S cell. Exp Hematol 17:263, 1989

30. Bradley TR, Hodgson GS, Rosendaal M: The effect of oxy-

gen tension on haemopoietic and fibroblast cell proliferation in

vitro. J Cell Physiol 97517, 1978

3

1.

Rich IN, Kubanek B: The effect of reduced oxygen tension

on colony formation of erythropoietic cells n vitro. Br J Haematol

52579, 1982

32. Ishikawa

Y,

Ito T: Kinetics of hemopoietic stem cells in a

hypoxic cultures. Eur J Haematol40:126, 1988

33. Lu L, Broxmeyer H E Comparative influences of phytohe-

moagglutinin-stimulated leukocyte conditioned medium, hemin,

prostaglandin E, and low oxygen tension on colony formation by

erythroid progenitor cells in normal human bone marrow. Exp He-

matol 13:989, 1985

34. Broxmeyer HE, Cooper S,Lu L, Miller ME, Langfeld CD,

Ralph P: Enhanced stimulation of human bone marrow macro-

phage colony formation in vitro by recombinant human macro-

phage colony-stimulating actor in agarose medium and at low oxy-

gen tension. Blood 76323, 1990

35. Johnson LV, Walsh ML, Bockus BJ, Chen LB: Monitoring

of relative mitochondrial membrane potential in living cells by fluo-

rescence microscopy. J Cell Biol 88:526, 1981

36. Bertoncello I, Hodgson

GS

Bradley T R Multiparameter

analysis of transplantable hemopoietic stem cells:

I.

The separation

and enrichment of stem cells homing to marrow and spleen on the

basis of rhodamine- 123 fluorescence. Exp Hematol 13:999, 1985

37. Hodgson GS, Bradley T R In vivo kinetic status of hemato-

poietic stem and progenitor cells as inferred from labeling with bro-

modeoxyuridine. Exp Hematol 12583, 1984

38. Lin H, Hsu

S:

Modulation of tissue mononuclear phagocyte

clonal growth by oxygen and antioxidant enzymes. Exp Hematol

14:840, 1986

39. Balin AK, Fisher AJ, Carter DM: Oxygen modulates growth

of human cells at physiologic partial pressures. J Exp Med 160: 152,

1984

40. Spangrude GJ, Johnson G R Resting and activated subsets

of

mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci

USA 87:7433, 1990

41. Visser JWM, van Bekkum DW: Purification of pluripotent

hemopoietic stem cells: Past and present. Exp Hematol 18:248,

1990

42. Oberley LW, Oberley TD, Buettner GR: Cell differentiation

and cancer: The possible role of superoxide and superoxide dismu-

tases. Med Hypotheses 6:249, 1980

43. Potten CS, Schofield R, Lajtha LG:

A

comparison of cell

replacement in bone marrow, testis and three regions of surface

epithelium. Biochim Biophys Acta 561:28 1, 1979

blood 1993 cipolleschi 2031 7

Documents