1. No category

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In Vitro Growth and Regulation of Bone Marrow Enriched CD34’ Hematopoietic
Progenitors in Diamond-Blackfan Anemia
By G.P. Bagnara, G. Zauli, L. Vitale, P. Rosito, V. Vecchi, G. Paolucci, G.C. Avanzi, U. Ramenghi, F. Timeus, and V. Gabutti
Diamond-Blackfan anemia (DBA) is a congenital red blood
cell aplasia. No clear explanation has been given of its
defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the
nature of this defect we studied the effect of several human
recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We
observed a defect underlying the early erythroid progenitors,
which were unresponsiveto several growth factors (erythropoietin, interleukin-3 [IL-31, IL-6, granulocyte-macrophage
colony-stimulating factor [GM-CSF], erythroid potentiating
activity), either alone or in association. The production of
cytokines was not impaired, and high levels of IL-3 and
GM-CSF were found in phytohemagglutinin-leukocyteconditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic
stem cells in DBA patients may be induced to differentiate to
the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34’ cell preincubation with IL-3.
Addition of the stem cell factor to IL-3 and erythropoietin
induces a dramatic in vitro increase in both the number and
the size of BFU-E, which also display a normal morphologic
terminal differentiation.
o 1991 by The American Society of Hematology.
D
ences in the experimental methods. In almost all reports,
the bone marrow population was not purged of accessory
cells, such as T, B, natural killer (NK) lymphocytes, or
adherent cells whose involvement in the control and production of stimulating factors was crucial; moreover, recombinant human growth factorswere used in only a few instances.
This article reports the effect of human recombinant
growth factors on a purified CD34’ population obtained
from the bone marrow of 10 DBA patients. Early erythroid
progenitors failed to respond to Epo, IL-3, IL-6, GM-CSF,
and erythroid potentiating activity (EPA), whether alone or
in association. The defect stems from the inability of
multipotent progenitors or CFU-GEMM (colony forming
units: granulocyte, erythroid, monocyte, and megakaryocyte) to undergo erythroid differentiation because proliferative and differentiative activity toward granulocyte, macrophage, and megakaryocyte lineages is normal. IL-3 and
GM-CSF production by peripheral mononuclear blood
cells was increased in all patients. The addition of stem cell
factor (SCF)”-’’ to IL-3 and Epo induced a significant
increase of the number and size of BFU-E both in normal
and in DBA cases.
IAMOND-BLACKFAN anemia (DBA) is a congenital red blood cell (RBC) aplasia characterized by
severe normochromic-macrocytic anemia appearing in early
infancy, often associated with phenotype abnormalities.
The bone marrow is normocellular, with a selective deficiency of RBC precursors or, in some cases, their arrested
maturation at the early stages of differentiation. The other
cell lineages are normal.
The exact explanation for this defective erythropoiesis
has not been firmly established, and several humoral or
cellular inhibitory factors have been proposed.
The erythropoietin (Epo) level in DBA is generally high.
Epo structure and function are normal, because the serum
can support the growth of normal bone marrow erythroid
progenitors or burst-forming units (BFU-E). Antibodies
against Epo have never been detected.14
The number of bone marrow erythroid progenitors, as
evaluated by in vitro growth of colony-forming units erythroid (CFU-E) and BFU-E, has been found to be defective
both at diagnosis and throughout the disease.”’
Although the erythroid lineage seems to be the only one
involved, earlier hematopoietic progenitors could be impaired, as moderate neutropenia or trombocytopenia may
occur later and a higher risk of progression to leukemia has
been documented.’
Cellular inhibition of erythropoiesis has sometimes been
observed: T lymphocytes from DBA patients inhibited
CFU-E growth when cocultured with normal bone marrow
cells, and adherent-cell or T-cell depletion may improve
erythroid progenitor growth.’ The meaning of these
studies is controversial and their results have not been
~0nfirmed.I~
l4
Recently, the purification of progenitor cells from lymphocytes and monocytes has improved the growth of erythroid
progenitors in patients responsive to steroids, but not in
resistant cases.’
It has also been shown that recombinant human interleukin-3 (IL-3) is capable of supporting in vitro erythroid
progenitor growth in some, but not all, DBA patients.“
Therapy with IL-3 or granulocyte-macrophage colonystimulating factor (GM-CSF) has also been experimented
with transient effect.I6
This lack of uniform data may be attributable to the
manifold phenotypes of DBA. It is primarily due to differBlood, Vol78, No 9 (November 1). 1991: pp 2203-2210
MATERIALS AND METHODS
Patients and methods. Bone marrow samples were obtained
from 10 DBA patients and 12 normal controls of the same age after
From the Institute of Histology and General Embryology, “Giop‘o
Prodi” Interdepartmental Centerfor Cancer Research, and the Department of Pediatrics III, University of Bologna; the Department of
Biomedical Sciences and Human Oncology and the Department of
Pediatrics, University of Turin, Italy.
Submitted October 1. 1990; accepted June 21, 1991.
Supported in part by an Italian Ministry of University and Scientific
and Technological Research Grant, and A.I.R.C. grants to Dr L.
Pegoraro and G.P.B.
Address reprint requests to Gian Paolo Bagnara, MD, Institute of
Histology and General Embryology, University of Bologna, Via Belmeloro 8, Bologna, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971I91 17809-0032$3.00/0
2203
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2204
BAGNARA ET AL
informed consent. Bone marrow aspirates were taken from controls for suspected hematologic disease not confirmed by subsequent analysis.
Peripheral blood samples (15 mL) were collected from these
patients and 10 normal donors to prepare phytohemoagglutininleukocyte-conditioned medium (PHA-LCM).
The patients were four boys and six girls, ages 2 months to 16
years (mean 7 2 5 years), all diagnosed within the first 5 months of
life (except for one at 2 years) on the basis of anemia with low
reticulocyte count and marrow erythroid hypoplasia (erythroid
nucleated cells 0.5% to 16.8%, mean 5.1% f 4.9%). In case 8,
maturation had been arrested at the proerythroblast level. Clinical
and hematologic data at diagnosis and the time of the study are
summarized in Table 1. All patients were studied at least 6 months
after suspension of steroid therapy or before its commencement
(cases 5 and 8). Serum Epo levels were higher than expected for
the current hemoglobin (Hb) level (mean Epo 448 f 254 mUimL,
mean Hb 9.4 ? 0.77 gidL).
Three patients presented congenital abnormalities (anophthalmus, ventricular septal defect, ventricular septal defect, plus
pulmonary valvular stenosis).
Recombinant growth factors. Recombinant human (rh) GMCSF, rhIL-6, and rhIL-3 were kindly supplied by Behring (Behring,
Marburg, Germany) and stored at -70°C for no more than 3
months before use. Their specific biologic activity was determined
by evaluating the number of granulocyte-macrophage colonies
(CFU-GM) obtained from normal bone marrow mononuclear
nonadherent cells (MNAC). rhEpo was a gift from Cilag Chemi
(Cologno Monzese, Milan, Italy). Human EPA was kindly provided
by Dr D. Golde (UCLA, Los Angeles, CA).*’As a source of SCF,
we used the media conditioned by COS cells transfected with an
expression plasmid containing cDNA encoding murine kit ligand
truncated 5’ to the transmembrane domain.” The SCF was kindly
supplied by Dr S.C. Clark (Genetics Institute, Cambridge, MA).
The dilution 1:200 gives the half-maximal activity, as measured by
the M-07E proliferation assay.
Cell separations. Heparinized bone marrow and peripheral
blood samples were diluted 1:2 with Iscove’s Modified Dulbecco’s
Medium (IMDM; GIBCO, Grand Island, NY), layered over
Ficoll-Hypaque (1077 sd; Pharmacia, Uppsala, Sweden) and centrifuged at 1,500 rpm for 30 minutes. Light-density mononuclear cells
(LD-MC) were collected and washed twice in IMDM supplemented with 10% of fetal calf serum (FCS). Mononuclear adherent
cells were then removed by two steps of incubation (1 hour) in
plastic flasks at 37°C in a humidified atmosphere of 5% CO, in air.
Subsequently, T-lymphocyte-depleted MNAC were obtained by
two steps of rosetting with neuraminidase-treated sheep RBCs.
After removal of T cells, a positive reaction to the OK T11 (CD2)
monoclonal antibody (MoAb) was constantly less than 1%. In cases
3, 9 and 10, the lymphocytes were negatively removed with
immunomagnetic beads coated with antimouse IgG (M-450 Dynabeads; Dynal, Oslo, Norway) after incubation at 4°C with the
following antibodies: CD2, CD19, CD22, CD14, CD56, and CD57.
Positive selection of CD34’ cells in bone marrow samples. Bone
marrow non-T MNAC, 5 x 105, were treated with 5 pL of My10
MoAb (CD34 Tecnogenetics, Milan, Italy) for 1 hour at 4”C, in a
final volume of 150 to 200 KL of phosphate-buffered saline
(PBS) + 1% FCS, under continuous shaking.
After two washings to eliminate the excess MoAb, cells were
treated with immunomagnetic beads coated with antimouse IgG
for 30 minutes in ice. A ratio of 3 to 4 beads per target cell was
found to provide the best recovery. CD34+cells were then collected
by a magnet (MPC 1, Dynabeads; Dynal) and resuspended in
IMDM + 10% FCS. After overnight incubation, they were washed
and gently pipetted to facilitate their separation from the beads;
60% to 80% of the total CD34’ population was recovered. The
fraction lost was mainly comprised of dead cells or cells coated too
heavily with beads. CD34’ cells mainly consisted of morphologically unidentifiable blast elements on May-Griinwald-Giemsa
staining, slightly contaminated by promyelocytes. In some
cases, the phenotype profile was performed after positive selection, with a nondetectable reactivity (constantly under 1%) to
Table 1. Hematologic and Clinical Data
1
Patients
At presentation
Sex
Age (mo)
Congenital anomalies*
Hb g/dL
Reticulocytes YO
WBC x 1 0 9 / ~
Platelets x 109/L
Bone marrow YOerythroid nucleated
cellst
Epo mU/mL
G.M
F
2
No
3.5
0.2
6.8
250
8
C.S.
P.A.
10
S.P.
M
M
F
440
1.5
Yes
4.6
0.1
11.4
492
1
No
7.6
0.1
7.2
330
5
No
5.6
0.1
8.5
320
0.4
380
16.5
400
1.8
350
-
7
10.3
0.5
4.7
36
Yes
Yes
169
10
8.3
0.2
6.4
384
Yes
Yes
686
2
L.D.
3
T.M.
4
C.E.
5
6
7
C.A.
M.S.
T.L.
F
5
Yes
6.8
0.3
13
200
M
24
No
3
0.2
3.5
200
F
F
2
No
3.2
0.3
9.9
150
2
No
2.4
0.1
9.9
450
F
2
No
2.3
0
10.2
200
M
1
Yes
6.6
0.2
9.7
1.5
5
5
9
5
6
-
-
-
-
-
-
16
9.5
0.2
4.5
220
Yes
No
600
5
9
0.2
5.8
230
Yes
No
193
4
10.5
0.1
6.7
320
Yes
No
822
7
10
0.2
8.2
300
Yes
No
349
2 (mo)
9.8
0.1
7.2
250
No
5
9
0.5
At the moment of study
Age (vr)
Hb g/dL
Reticulocytes YO
WBC x 1 0 9 / ~
Platelets x 109/L
Regular transfusion therapy
Steroid response
Epo mU/mL
*Congenital anomalies: ventricular septal defect patient 2, ventricular septal defect
tBM YOerythroid cells: patient 8 maturative arrest at the proerythroblast level.
-
8.3
0.1
7.3
150
Yes
No
11
9.1
0.05
4.0
231
Yes
No
-
-
-
7 (mol
8.9
0.05
12.8
40 1
No
320
+ pulmonary stenosis patient 8, anophthalmus patient 7.
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2205
REGULATION OF HEMATOPOIESIS IN DBA
CD4, CD2, CD16, and CD19 (Ortho Diagnostic System, Raritan,
NJ).
Cell culture. The colony assay for BFU-E was performed
according to Iscove et al.” Bone marrow CD34+ cells, lo4, resuspended in 0.1 mL of PBS + 1% detoxified bovine serum albumin
(BSA, Fraction V Chon; Sigma, St Louis, MO) were plated in a
1-mL mixture of IMDM containing 30% FCS, 2 X
mol/L
hemin, 5 x lo-’ mol/L p-mercaptoethanol, 1% BSA, and 0.9%
methylcellulose. The cells were stimulated, in our optimal standard
conditions, with rhEpo 2 U and rhIL-3 100 U or with various
combinations of recombinant growth factors, as specified below.
The assay for the 14-day CFU-GM was performed as previously
described.24CD34’ bone marrow cells, lo4,were plated in 35-mm
Petri dishes in 1 mL of IMDM containing 20% heat-inactivated
FCS, 0.3% noble agar, and 200 U rhGM-CSF + 100 U of rhIL-3 as
the standard source of colony-stimulating activity.
For the megakaryocyte colony-forming units (CFU-MK) assay,
plasma clot assay was performed according to McLeod et ala and
Vainchenker et al.” CD34’ cells, 104, were seeded in IMDM,
supplemented with 20 pg L-Asparagine (Sigma) 3.4 pg CaCI,, 10%
BSA, 10% of a preselected batch of heat-inactivated pooled human
AB serum, and 200 U rhGM-CSF. After 12 days of incubation, the
plasma clot was fixed in situ with methanol-acetone 1:3 for 20
minutes, washed with PBS and double-distilled water, and then
air-dried. Fixed plates were stored at -20°C until immunofluorescence staining was performed. CFU-MK colonies were scored as
aggregates of 3 to 100 cells intensively fluorescent to J15 (CD41W)
MoAb directed against the IIB-IIIA glycoprotein complex. Binding
was shown by fluorescinated goat antimouse IgG (Ortho).
To clarify whether different combinations of recombinant factors influence bone marrow progenitors, and especially BFU-E
growth in DBA patients, we tested the effect of increasing
concentrations of Epo 2 to 6 UimL alone or in combination with:
IL-6 (1 to 100 UimL), GM-CSF (200 UlmL), IL-3 (100 UimL),
EPA 1:1,OOO, IL-3 (100 U) PIUS GM-CSF (200 U), IL-3 (100 U)
plus IL-6 (1 to 100 U/mL).
In three patients (cases 3,9, and 10) and in three normal controls
the effect of the SCF on BFU-E growth was studied on the CD34’
positive bone marrow population. The SCF has been tested at
1:100, L:200, and 1:400 in association with Epo 2 and 4 U/mL, and
IL-3 (100 UimL).
Pre-exposure experiments in liquid cultures. CD34t cells from
four patients and four controls were either seeded immediately in
semisolid media or preincubated in IMDM + 10% FCS in the
presence of rhIL-3 (100 UlmL). Daily (up to the fourth day)
aliquots were collected for clonogenic assays. The percentage of
CD34’ cells was determined by immunofluorescence. BFU-E,
CFU-GM, and CFU-MK growth is expressed as the number of
colonies by lo4CD34+ cells.
Preparation of PHA-LCM. LD-MC from the peripheral blood
of the 10 DBA patients and 10 normal donors were seeded in
IMDM, supplemented with 10% FCS and 1% phytohemoagglutinin (PHA) at a concentration of lo6cellsiml, and incubated for 72
hours. The conditioned medium was than harvested, centrifuged
for 10 minutes at 2,000 rpm, filtered, and stored at -70°C for no
more than 3 months, before use on the M-07E cell line.
Anti-GM-CSF and anti-K-3. Murine MoAb anti-GM-CSF
and anti-IL-3 were purchased from Genzyme Co (Cambridge,
MA). A fixed dilution of 1:100 (PBS 1% FCS) was tested on
0.005% to 10% vol/vol concentrations of both normal and DBA
patients’ PHA-LCM.
Biologic assay for the production of GM-CSF and IL-3. The
GM-CSF and IL-3-dependent M-07E cell line2’ was kindly provided by Dr L. Pegoraro (University of Turin, Italy). PHA-LCM
was used as a source of growth factors at a final concentration of
+
1% for 96 hours. Exponentially growing M-07E cells were washed
twice and seeded at 1 x 106cellsimlin IMDM with 5% FCS. After
24 hours of IL-3 deprivation, 5 x lo4cells were washed twice and
seeded in 200-pL microwells in IMDM with 5% FCS in the
presence of increasing concentrations of PHA-LCM (from 0.005%
to 10% vol/vol). After 28 hours, triplicate wells were pulse-labeled
with ’H-TdR (1 pCi/well) for 4 hours. Alcohol acid precipitated
radioactivity was determined by liquid scintillation counting.
The concentration of IL-3 and GM-CSF in PHA-LCM was
determined by comparing the dose-response curves obtained with
serial dilutions of rhIL-3 and rhGM-CSF and of PHA-LClk
GM-CSF and IL-3 levels were determined by subtraction of the
number of counts per minute (cpm) obtained in the presence of the
anti-IL-3 or anti-GM-CSF MoAb from the total cpm amount.
Epo measurement. Serum Epo levels were determined by raditlimmunoassay= using radiolabeled hormone and anti-Epo antibodies (Ingstar; Sorin, Saluggia, Italy).
Statistical analysk. The results were expressed as means f 1
standard error of mean (SEM) of the data from two or more
experiments performed in duplicate. Statistical analysis was per.
formed using the two-tailed Student’s t-test, linear regression, and
correlation tests.
RESULTS
In vitro growth of enriched (CD34+) bone marrow cells.
The results of the in vitro growth of 14th day CFU-Gd,
CFU-MK, and BFU-E in our standard culture conditions
are summarized in Table 2.
Our assay did not assess CFU-E growth. Culture of
CD34’ cells does not give rise to CFU-E colonies at day 7
because they are earlier progenitors, and only the 14th da$
BFU-E are detectable.
The number of CFU-GM and CFU-MK colonies derived
from DBA marrows was similar to that from normdl
controls (P > .05). In contrast, a dramatic decrease in tHe
erythroid progenitors was observed in 9 of 10 DBA patients
(P < .01).
Production of IL-3 and GM-CSF fromPHA-LCM of DBA
patients. We investigated whether production of the cytdkines involved in erythroid differentiation accounts for
impaired erythropoiesis in DBA patients. The production
of IL-3 and GM-CSF by DBA patient and normal contrdl
Table 2. In Vitro Growth of Granulomacrophage(CFU-GM),
Megakaryocyte(CFU-MK),and Erythroid (BFU-E) Progenitors From
10’ CD34‘ Enriched Bone Marrow Samples
Case No. DBA
1
2
3
4
5
6
7
8
9
10
Mean 2 SEM
Controls
(mean of 12 cases
CFU-GM
CFU-MK
BFU-E
20
13
16
23
27
28
25
18
22
16
10
35
32
23
17
18
16
20.7 ? 2.4
2
7
3
4 t 2.3
23.2 t 16
62 z 14
a
9
20
18.9 -+ 2.2
lr
SEM)
29
* 6.5
0
0
2
1
2
1
25
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2206
BAGNARA ET AL
peripheral blood mononuclear cells was tested on the
M-07E cell line.
Addition of 1:lOO vol/vol anti-GM-CSF and anti-IL-3
completely abrogated the stimulatory activity of 10 pg/mL
IL-3 and 2 to 3 pg/mL GM-CSF, respectively (data not
shown). However, in the experiments performed on PHALCM, incomplete neutralization with anti-GM-CSF and
anti-IL-3 MoAbs was observed at the higher PHA-LCM
concentrations (1% and 10%) (Fig 1).This finding may be
partially explained by the presence of other cytokines (ie,
IL-2, IL-4, and IL-9) to which the M-07E line is sensitive,
although not dependent on them.2Y
The amount of both IL-3 and GM-CSF was significantly
higher (P < .01) in DBA patients: 384 ~f:27 pg IL-3 and
302 f. 12 pg GM-CSF versus 206 -r- 15 pg and 230 ~f:9.2 pg
in normal controls. The difference in IL-3 production was
statistically significant at the final PHA-LCM concentration
of 10% (P = .034), whereas that in GM-CSF levels was only
significant at 0.5% (P= .001) and 0.1% (P = .003), indicating that IL-3 production is more pronounced than that of
GM-CSF in DBA.
I l l
0
0.01
0.1
10
% of PHA-LCM
40000
T
0
0.01
0.1
1
IO
X of PHA-LU1
Fig 1. Biologic assay on the M-07E cell line for the production of
GM-CSF and IL-3 by PHA-LCM obtained from 10 DBA patients (A) and
10 controls (B). Data are the means f SEM of three separate
controls; (-+-),
experiments performed in duplicate. (A) (-m-),
anti-GM-CSF; (-0-),
anti-IL-3. [B) (-El-),
DBA patients; (-+-),
anti4WCSF; (-0-),
anti-IL-3.
/1 /1
200 -
DBA patients
--t
Controls
100 -
x
0
. & .
0
=
,-.
20
.
I
X
I
6
Y
.-
40
60
80
100
TIHE o f LIWID CULTURE (hours)
Fig 2. BFU-E growth after pre-exposure to IL-3. CD34‘ cells from
four DBA patientsand four normal controls were pre-exposed in liquid
culture to 100 U of rhlL-3. After 12, 24, 48, 72, and 96 hours, CD34’
cells were harvested and seeded in methylcellulosemedium for BFU-E
growth. Data represent the means 2 SEM of the number of colonies
observed in four separate experiments. Each point was performed in
duplicate.
Re-exposure to ZL-3. Exposure to IL-3 increases the
number of all the hematopoietic progenitors and induces
their differentiati~n.~’
Therefore, we investigated the effect
of a 12- to 96-hour exposure of bone marrow CD34+ cells to
100 U of IL-3 in liquid cultures in DBA and in controls.
After 96 hours in controls, the number of BFU-E was
twofold to threefold higher than in non-pre-exposed cultures. The number of BFU-E was constantly low in DBA
patients (Fig 2). CFU-MK and CFU-GM were significantly
increased after 96 hours and their growth pattern was
similar in both DBA patients and normal controls (Fig 3).
Effect of cytokines on the in vitro growth of BFU-E. The
effect of different combinations of recombinant factors on
the growth of control and DBA BFU-E is illustrated in
Table 3. Hemoglobinized erythroid colonies were never
detected in the absence of Epo. In CD34’ enriched normal
bone marrow cultures, these combinations increased the
number of BFU-E compared with Epo alone, peak stimulation being achieved by Epo (2 U) + IL-3 (100 U). DBA
BFU-E were never increased.
The response of the erythroid progenitors to different
dilutions of SCF in three DBA cases and in normal controls
is illustrated in Table 4. Addition of even the lowest SCF
dilution to Epo and IL-3 induced a dramatic increase in the
number of both normal and DBA BFU-E. This finding was
the same when Epo was increased from 2 to 4 U/mL (data
not shown). There was a striking increase of the size of each
aggregate in a single BFU-E (from a mean of 30 cells to
more than 200 cells) (Fig 4).
DISCUSSION
Several reasons have been proposed for the erythropoietic inhibition observed in D B A a humoral mechanism
involving autoimmunity or the presence of inhibiting factors,3’,’’ a cellular mechanism mediated by T lymphocytes,
NK cells, or adherent cells,”” impaired cytokine production
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2207
REGULATION OF HEMATOPOIESIS IN DBA
Table 4. Number of BFU-E/ 10' CD34' Bone Marrow Cells From
Three DBA Patientsand Three Normal Controls After Stimulation
With Epo (2 U) IL-3 (100 U) or Epo (2 U) + IL-3 (100 U) + SCF at
Three vol/vol Dilutions
+
T
T T ~
DBA Cases
Case 9
Case 10
Controls
0
0
4
34 2 2.0
32
34
33
28
24
25
36
30
36
116 8.4
81 2 14.2
96 f 13.1
Case 3
+
+
IL-3 100 U EPO 2 U
IL-3 100 U EPO 2 U
SCF 1:lOO
1:200
1:400
+
*
The dilution 1:200 gives the half-maximal activity on M-07E proliferation; the same half-maximal stimulation on M-07E is obtained by the
addition of 5-10 ng/mL rhSCF (Santoli D, et al, manuscript in preparation).
O h
96 h
PRE--D(POSITION TO 100 U of
IL-3 IN LIQUID CULTURES
Fig 3. CFU-GM and CFU-MK growth in agar and plasma clot media.
CD34' cells from four DBA patients and four normal controls were
pre-exposed to IL-3 for 96 hours. Data are the means e SEM of four
separate experiments. Each point was performed in duplicate. (El),
CFU-GM DBA; (W), CFU-GM controls; (E),CFU-MK DBA; (0).
CFU-MK
controls.
or function, or an intrinsic defect within the hematopoietic
stem cells.'~'
A decrease in bone marrow BFU-E and CFU-E number
in DBA has been observed?.' In contrast, attempts to
enhance erythroid progenitor growth and differentiation
have given conflicting results. The growth defect can be
partially corrected in vitro by addition of high doses of Epo
or IL-3 or ~ter0ids.l~
Interestingly, the impairment of in vivo erythropoiesis
seems to be more dramatic in DBA patients who do not
respond to steroid the rap^."^
An increased number of reticulocytes and a decreased
blood requirement in some transfusion-dependent and
steroid-unresponsive DBA patients have been recently
Table 3. Effect of Different Combinations of Growth Factors on the
In Vitro Growth of Erythroid Progenitors (BFU-E) From Enriched
Bone Marrow Samples (WCD34' cells/plate) of 10 DBA Patients
and 12 Normal Controls
~
Epo 2 U
Epo 4 U
Epo 6 U
EPO2 U+ IL-3 100 U
EPO2 U+ IL-3 100 U + IL-6 1 U
EPO2 U+ IL-3 100 U IL-6 10 U
EPO 2 U+ IL-3 100 U + IL-6 100 U
EPO 2 U+ IL-6 1 U
EPO 2 U+ IL-6 10 U
Epo 2 U+ IL-6 100 U
EPO2 U+ GM-CSF 200 U
EPO2 U+ GM-CSF 200 U + IL-3 100 U
Epo 2 U+ EPA 1:lOO
+
Controls
DBA
26 f 7
30 2 13
32 2 13
62 f 14
45 13
46 f 14
60 2 15
20 6
19 f 6
19 f 7
4 5 2 13
60 f 14
55 f 14
1.2 0.7
1.3 f 0.6
2.1 f 1.1
4.3 f 2.3
4.5 f 2.6
3.4 f 1.9
3.6f 2
1.5 f 0.8
1.6 0.8
1.1 0.4
3.6 f 1.1
3.6 1.8
3.5 2 1.4
*
*
*
*
Data are expressed as means f 1 SEM. Each point was performed in
triplicate.
reported following the administration of GM-CSF and
IL-3.I6
All these findings suggest a possibility of improving
erythroid growth. The results obtained have always been
scanty and transient. Although this could be due to differences in the genetic defect, patient's age and clinical
history, it must be underlined that the bone marrow progenitors have not always been concentrated and purified
from accessory cells, and recombinant growth factors were
not used by some workers.
In our experiments, only CD34' positively selected progenitors, virtually free from accessory cells, were plated in
the presence of well-defined recombinant growth factors.
We also investigated the ability of patient's mononuclear
cells to produce GM-CSF and IL-3, which are well-known
regulators of erythropoiesis.
Our observation that addition of various recombinant
cytokines to a CD34' positively selected lymphocyte and
monocyte-free marrow population always fails to support
the growth of BFU-E in DBA challenges the supposition of
a direct lymphocyte or monocyte involvement in the BFU-E
growth defect.
The inhibition of erythropoiesis reported by some investigators in cocultures with T lymphocytes from DBA patients
may be ascribable to an immunologic phenomenon related
to HLA antigens in subjects regularly t r a n s f ~ s e d . ~ ~
After pre-exposure to IL-3, hematopoietic progenitors
were induced to differentiate to the granulocyte and the
megakaryocyte, but not the erythropoietic lineage. This
finding indicates that the low number of BFU-E is not due
to a diminished population of multipotent stem cells, but to
a defect in the passage from the CFU-GEMM to BFU-E as
shown by the lack of progenitor response to the in vitro
stimulating activity of Epo and IL-3, IL-6, GM-CSF, EPA,
alone or in association.
Moreover, impaired production of these cytokines in
DBA is denied in our experiments. In a highly sensitive
biologic assay, PHA-LCM produced increased amounts of
IL-3 and, to a lesser extent, GM-CSF. However, if we take
into account the central role played by IL-3 and by
GM-CSF in normal bone marrow BFU-E growth and
differentiation, it is conceivable that these increases, along
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
2208
BAGNARA ET AL
A
Fig 4. Size of erythroid aggregates from DBA
CD34' bone marrow cells. (A) Cultures stimulated
with Epo (2 UlmL) + IL-3 (100 UlmL): (8)cultures
stimulatedwith Epo (2 UlmL) I IL-3 (lo0 UlmL) and
SCF 1:200vol/vol.
with thc wcll-dtxumcntcd clcviition of scriim Epo in DRA.
arc an attcmpt to compcnsatc for crythropoictic fiiilurc.
Thc in vivo cytokinc network is still poorly tlcfincd ant1
othcr factors may hc involvctl in stem ccll expansion and
erythroid diffcrcnti:ition. Thc rcccntly tlcscrihcd hcniatopoictic SCF (;ilso callcd mast ccll growth fwtor. stccl locus
factor. c-kit ligand) has hccn shown to intlucc an incrcilsctl
prolifcration of hcmatopoictic propnitors and to display :I
potent svncrgistic activitv in combination with II.-2 m d
othcr cytokincs.'-'' SCF also sccms to play ;in importiint
rolc in thc ;ictiv;ition of primitivc cclls. while tlic dil~crcntiation signal is prrwidcd hv thc spccific C'SFs. Morccwcr. its in
vivo administration rcvcrscs anemia in SI /SItI nititant mice."
In our cxpcrimcnts, in vitro ;idtlition of SCF to 1L-3and
Epo intluccd ;I signifkint incrcasc of thc numhcr and sizc
of both normal iintl DRA DFU-E. This cffcct was constant
and striking. Stimulation of DRA crythroitl progcnitors was
obccnrd i i t thc SCF conccntration r a n g that provides
optimal growth cnh;inccmcnt o n normal CD34' cclls and
M-07E cells (S.C. Clark. unpuhlishctl data), suggesting that
in DRA. SCF rcccptor is normally cxprcsscd o n SCFrcsponsivc CI1.14' cclls. Thc hypothesis that othcr rcccptor
molecules. such ;IS Epo rcccptors. arc involvcd in thc
tlisc;isc can not hc ruled out. The ability of SCF to inducc
DFU-E proliferation ;ind dilicrcntiation in DRA indiciitcs a
similwity hctwccn this discmc and thc miirinc complcx
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
2209
REGULATION OF HEMATOPOIESIS IN DBA
hematopoietic defect caused by mutations of S1 and W
gene^.'^
In conclusion, our findings show that SCF significantly
reverses the erythropoietic impairment in DBA in vitro,
suggesting a possible therapeutic utility in vivo.
Further studies are needed to see whether the inability of
the bone marrow to produce or release a biologically active
form of this factor is involved in the pathogenesis of DBA.
ACKNOWLEDGMENT
We thank Dr S.C. Clark for kindly providing SCF and Dr L.
Pegoraro for helpful discussion.
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1991 78: 2203-2210
In vitro growth and regulation of bone marrow enriched CD34+
hematopoietic progenitors in Diamond-Blackfan anemia
GP Bagnara, G Zauli, L Vitale, P Rosito, V Vecchi, G Paolucci, GC Avanzi, U Ramenghi, F Timeus
and V Gabutti
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