human bone marrow Characterization of a bipotent



human bone marrow Characterization of a bipotent
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1996 88: 1284-1296
Characterization of a bipotent erythro-megakaryocytic progenitor in
human bone marrow
N Debili, L Coulombel, L Croisille, A Katz, J Guichard, J Breton-Gorius and W Vainchenker
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Characterization of a Bipotent Erythro-Megakaryocytic Progenitor i n
Human Bone Marrow
By Najet Debili, Laure Coulombel, Laure Croisille, Andre Katz, Josette Guichard, Janine Breton-Gorius,
and William Vainchenker
The aim of the present study wast o determine if the human ficiency of CFU-MK and ofBFU-E/MK progenitors when comerythroid (E)and megakaryocytic (MK)lineages were closely
bined with SCF, IL-3. and Epo. In contrast, when these cullinked to the
existence of a bipotent burst-forming unit
in the presence of 30% fetal calf
E/MK progenitor.In methylcellulose cultures, BFU-E/MK colserum, no BFU-E/MK colonies were observed irrespective
onies were observed at day 12 and closely resembled mature
of the combination of growth factors used, including the
component was
BFU-E with the exception that the erythroid
presence of MGDF; however, inclusion of the MS-5 cell line
surrounded by MK. These colonies were quitedifferent from
restored the growth of this bipotent
progenitor. In contrast,
the colony forming unit (CFU)-GEMM-derived colonies,
in cultures performed in the presence of human normal or
which were composed of a larger number of erythroblasts
aplastic plasma, MS-5 had only a slight effect on the cloning
and whichdeveloped later in culture. The existence of these
efficiency but improved MKcytoplasmic maturation and MK
bilineage colonies composed of 100 t o 1,000 erythroblasts
size, suggesting that the maineffect of MS-5 is t o diminish
intermingled with a few MK and without
granulocytic cells
the inhibitoryeffect of the fetalcalf serum on theMK differwas confirmed by theplasma clot technique and immunoentiation. The clonal origin of bipotent BFU-E/MK colonies
alkaline phosphatase labeling of the MK. To investigate if
was demonstrated in liquid culture of single CD34' CD38'OW
this bipotent progenitor belonged t o t h e compartment of
cells by immunophenotyping individual clones. At day 12,
primitive progenitors, CD34' marrow cells were subfraction30% of the clones contained erythroblasts (glycophorin A')
ated according t o expression of the CD38 antigen. The bipoand some MK (CD41') without granulocytes ( G ) or macrotent BFU-E/MK progenitor as well as a large fraction of MK
phages (M) (CD14' and CD15'). A t day 20, clones containing
in the CD34+ CD38' or in theCD34'
progenitors were found
erythroblasts and MK were rare (5%). In contrast multilinCD38- cell fractions. Growth of this bipotent
BFU-E/MK proeage clones could be frequently detected at this time withgenitor required the combination of stem cell factor (SCF),
out passage from BFU-E/MK clones at day 12 t o GEMM at
interleukin-3 (IL-3). and Epo in serum free conditions. Addiday 20. These resutts suggest that a bipotent BFU-E/MK
tion of IL-6 had only a marginal effect, whereas megakaryoprogenitor may bea nonrandom step in the hierarchical decyte growth and development factor (MGDF) was not an
velopment of stem cells.
absolute requirement, but slightly increased the plating efC 7996 by The American Society of Hematology.
HE HEMATOPOIETIC system is composed
of heterogenous populations of cells that have been schematically
divided into three
compartments,' ie, a stem cell,a progenitor
cell, and a terminally differentiating cell compartment. Regulation of cell proliferation and differentiation in the progenitor compartmentis mainly controlledby identified cytokines,
but molecular mechanisms involved in the commitment of
a pluripotent stem cell toward one celllineage are presently
unknown. Several lines of evidence, most of them based on
the analysisof thecomposition of colonyforming unit
(CFU)-Sor of the progeny of paired daughter
that decisions at the level of early stem cells are stochastic.
However, whether the restriction of stem cells potential occurs progressivelythrough the production of intermediate
oligopotent progenitors with all the various combinations of
potentialities or only after one division with the generation
of only monopotent progenitors
From INSERM U362, Insritut Gustave Roussy, Villqjuff; and
INSERM (191, Hopital Henri Mondor, CrPteil, France.
Submitted September IS. 199s; accepted April I I , 1996.
Supported by grants from the Institut National de la Sante' et de
la Recherche Me'dicale, Association de la recherche contre
le cancer.
Ligue nationate conare le cancer, and Electricif6 de France.
to NajetDebili,PhD, INSERM U.162.
Institut Gustave Roussv, Villejuih 94805, France.
The publication costs of this article were defrayedin part by page
chargepayment. This article must therefore he herebymarked
"advertisement" in accordance with 18 U.S.C. section 1734 soiel~to
indicate this fact.
0 1996 by The American Society of Hematolog?.
transfer experiments have demonstrated that a bipotent neutrophWmacrophage progenitor exists that retains hiresponsiveness toboth granulocyte macrophagecolony stimulating
factor (GM-CSF) and M-CSF.7 Theexistence of such a tight
linkage between two celllineagescould
be anexception
along the hematopoietic differentiation pathway, but it cannot be excluded that other types of bipotent progenitors also
exist. For several years, numerous similarities between the
erythroid (E) and megakaryocytic (MK) celllines have been
emphasized. In humans, almost all leukemiccelllines described as erythroleukemic or megakaryoblastic express Eand MK-specific genes and this dual expression is found in
the same cell.'" Similar observations have also been made
in some acute leukemias."' Indeed, it has been shown that
the regulation of E- and MK-specific genes share many features""' and transgenicmice expressing thymidinekinase
gene under the control of the GPIIb promoter have defective
erythropoiesis and megakaryocytopoiesis." It is thus possible that restriction toward the E or the MK pathways occurs
relatively late in the hematopoietic hierarchy and that a bipotent BFU-E/MK can be identified.15 Mixed E-MK colonies
have been described by several investigators in both humans
and mice,"-"' but no detailedanalysis of their properties have
beenprovided. The present experiments used clonogenic
assays and single cell cultures to document the existence of
human bone marrow cells able to generateonly erythroblasts
and megakaryocytes, to determine their phenotype, relative
frequency, andoptimal
growth requirements. This burst
forming unit (BFU)-E/MK segregated in the CD34'/CD38'""
cell fraction, required 12 days to differentiate and produced
exclusively erythroblasts and megakaryocytes.
Blood, Vol 88, NO 4 (August 151, 1996: pp 1284-1296
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HPCA-2 (CD34) and FITC-IOB6 (CD38). Mer one washing, cells
were suspended in IMDM at a concentration of 5 X [email protected]/&and
Bone Marrow Cells
separated by cell sorting. In other experiments, triple staining with RBone marrow was obtained from patients undergoing hip surgery PE-HPCA-2,FITC-anti-CD38,andincubationwithHoechst33342
after informed consent was obtainedin accordance with the institudye (Sigma ata concentration of 5 pg/mL for 90minutes at 37OC’’ was
tional guidelines of the Committee on Human Investigation. Cells
performed to study the cell cycle status of hematopoietic progenitors.
were collected by vigorous shaking of bone fragments in Iscove’s
Cells were sorted on anODAM,ATC 3000” cell sorter(ODAM/
modified Dulbecco’s medium (IMDM, GIBCO, Paisley, Scotland),
Bruker, Wissembourg, France)
or a FacsVantage (Becton Dickinson)
supplemented with 100 pg/& of deoxyribonuclease (DNase t y p
equipped with two argon ion lasers (INNOVA 70-4 and 90-5; CoherI; Sigma, St Louis, MO), centrifuged once, counted, and separated
ent Radiation, Palo Alto, CA) tuned to 488 and 360 nm, respectively,
and operating at 500 mW. Amorphologicgateincluding all the
(<1.077 g/mL) marrow cells (LDMC) were recovered and after
CD34’ cells wasdeterminedontwo-parameterhistograms(side
washing were used either for isolation of CD34+ cellsby the immuof thecellvolume
nomagnetic bead technique or flow cytometry.
[ODAM] or the forward Scatter [FSC, FacsVantage]). Compensation
for two-color labeled samples wasupsetwithsingle stained samples.
Negativity for the CD38 among the CD34+ cells was determined
using control cells labeled with the PE-HPCA-2 and an irrelevant
Fl’K-a~~ti-ClXl (anti-GpIIbm Immunotech,Luminy, Fran~e),
IgGl monoclonal antibody (MoAb).
FITC-anti-CD61 (GpIUa; Dako, Glostrup, Denmark), FITC-antiFor limiting dilution experiments, CD34+
CD38”” cells were diglycophorin A (GPA) (Dako), R-PE anti-GPA (Immunotech), FITCrectly sorted into 96-well tissue culture plates (Fklcon, France) with
an automatic cloning device unit.
Dako), FITC anti-CD38 (IOB6) (Immunotech),
FITC-CDllb (Mac1, granulocytic and monocytic differentiation, Immunotech), FITCAssessment of Hematopoietic Progenitor Cells
R-PEHPCA-2 (CD34; Becton Dickinson, Mountain View, CA), and RQuantitationofclonogenicprogenitorsinsemi-solidassays. EryPE anti-CD14 (Gp55, granulocytic and monocytic differentiation,
Becton Dickinson) were used either for cell sorting or analysis by
(CFU-GM), megakaryocytic (CFU-MK), and mixed(CFU-GE“)
flow cytometry. The unconjugated antibody Y2/51 (anti-CD61) was progenitors were quantified using previously described methylcellua generous gift from Dr D. Mason (Oxford, UK).
lose assays?’ Each cellular fraction selected was plated at a concentration of 0.5 to 2 X lo3 cells/mL of complete methylcellulose meIsolation of CD34 by the Immunomagnetic Bead
dium (0.8% methylcellulose in IMDM, 30% fetal calf serum
1% deionizedbovineserumalbumin
PSA] and iO-4 m o m 0Technique
mercaptoethanol). Cultures were also performed in the presence of
CD34+ cells were recovered from LDMC (2 X 10’ cells) by the
sera from aplastic patients or in serum-free medium as previously
immunomagnetic bead technique using the Dynal kit (Dynabeads
1.5%deionized senim
M450 CD34) according to the manufacturer’s instructions (Dynal,
albumin (Cohn fractionV Sigma ChemicalCO),iron saturated huOslo, Norway). CD34+ cells were detached from the beads using
man transferrin (300p g / a , Sigma), calcium chloride (28 ng/&),
the DETACHa BEAD solution (Dynal).
a mixture of sonicated lipids, 7.5X lO-4 mom, cy-thioglycerol, 100
ng/mL insulin, and 0.8% chemically pure methylcellulose in IMDM.
Cell Sorting
The lipid mixture was produced by sonicating 7.8 mg cholesterol,
Subpopulations of CD34+ cells were
sorted according to their expres- 6.14 mg oleic acid, and 7.4 mg dipalmitoyl lecithin (all obtained
sion of the CD38 antigen starting from LDMC
or magnetic bead isolated fromSigma)in 10 mL ofIMDM(withoutsodiumbicarbonate)
the R-PEcontaining 1%albumin. Aliquots (20 pUmL) of this mixture were
Fig 1. BFU-E/MK colonies in methylcellulose and plasma clot cultures. (A) A typical BFU-EIMK colony observed with aninverted microscope
in cultures containing 30% FCS and MS-5 cells. This type of colony contained erythroblasts and megakaryocytes. The other cells observed on
the figure correspond to MS-5 cells. (B) A similar colony in plasma clot. MK appeared in pink color after immunolabeling with an anti-CD61
MoAb. (C) A typical BFU-E/MK colony in cultures where FCS has been replaced by AP.
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Table 1. Numbers of Hematopoietic Progenitor Cells in the Different Cell Fractions Sorted According to Their Expression of CD34 and CD38
Antigens and Plated in Methylcellulose Colony Assays With Either FCS or AP
Progenitor Cells per 1,000 Cells
MK col
One thousand cells of the different subfractions of CD34' cells were plated in methycellulose colony assays in the presence of either 30%
FCS or 10% AP and SCF + IL-3 + Epo a s growth factorMS-5 cells were added at the concentration
of 10,000 cells per dish.ElMK were enumerated
in the mBFU-E and scored at day 10 to 12 and CFU-GEMM were enumerated in the iBFU and scored at days 20 to 25. Results are from one
experiment. Numbers refer to the meanof two counts obtained in two separate plates.
added to the culture medium. Colony-stimulating factors were pro10%
vided either as agar-leukocyte conditioned medium (agar-LCM:
vollvol) or as recombinant human (rh) growth factors: rhStem cell
factor (SCF) (S0 ng/mL). G-CSF (20 nglmL). and megakaryocyte
(MGDF) (10 nglmL)alsocalled
Mpl-ligand (Mpl-L), or thrombopoietin (TPO) (all three kindly provided by AMGEN, Thousand Oaks, CA), rhIL-6(100 U/mL). rhIL3 (100 UlmL), rhGM-CSF (2 ng/mL) (kindly provided by Immunex.
I UlmL;AmerSeattle, WA) andhumanerythropoietin(rhEpo:
sham). Petri disheswerecultured at 37°C in a S 8 C0?-9S% air
humidified incubator. Hematopoietic progenitors were scored at least
twice at days 12 to 14 and 20 to 25.
Cultures were also performed by the plasma clot technique using
platelet poor plasma (PPP) and a combination of cytokines." When
MGDF became available, cultures were performed
by the serumfree fibrin clot.'5 Briefly, ingredients for serum-free cultures were
the same as for methylcellulose cultures with the bovine citrated
by bovineplasma fibrinogen ( I mglmL.
Sigma), 0.01 m o l L e aminocaproicacid,andhorsethrombin
3834+ 38+ 38+
Exp 1
studied after 12 days. Colonies were quantifiedby an indirect immunophosphatasealkalinelabelingtechniqueusingananti-GplIIa
MoAb (CDhl, Y2-S I ). Dishes were scanned completely under an
inverted microscope at 40 or 100 X magnification.
We have previously reported that addition of murine stromal cells
from the MS-S cell line to hematopoietic colony assays selectively
stimulates the expression of very early clonogenic progenitor cells
(immature RFU-E. CFU-GEMM) in synergy with growth factors.'"
The MS-S stromal cell line" was kindly provided by K. Mori and
maintained in CYMEMsupplemented with 10% FCS. Its growthpromoting effect was assessed by adding 10.000 MS-S cells to both
methylcellulose and plasma clot assays.
Single cell cdnrres. In limiting dilution experiments. individual
CD34* CD38'"" weresortedinto96-wellplatesusingserum-free
medium and a combination of seven cytokines (SCF. IL-3. Epo. IL6 , G-CSF, GM-CSF. and MGDF) or of AP and six cytokines (all
except MGDF). Plates were examined
at day I I to 13. day 18 to
20. and later after incubation at 37°C in an air atmosphere supplemented with S 8 CO:.
Individual clones were cytocentrifuged or divided into two
for flow cytometric analysis. The first half was doubly labeled with
34 +
Exp 2
Fig 2. Phenotype of the BFU-E/MK progenitor.
This figure represents the results
of two representative experiments in which CD34' cells have been
subfractionated in CD38'. CD38'. and CD38- subfractions. Cultures were performed
in plasma clotin triplicate in the presence ofAP and the combination of
SCF, IL-3, and Epo. MK were identified as in Fig 1B.
CD38' or CD38- cells (lo3)and CD38' cells (5 x 10')
were plated. Results are expressed
a s t h enumber of
colonies per lo3 plated cells.
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Fig 3. Kineticsof development ofBFU-E,CFUMK, and BFU-EIMK colonies from different CD34'
cell subfractions. Experiments were designed as in
Fig 2 and cultures were studied at day 7, 12, and 18.
cell tractton W+
Ident$cation qf a Bipotent Enthroin-Megnkonoc?.tic
(BFU-ELWK) Progenitor Cell
In preliminary experimentsaimed at optimizingculture
conditions for the growth ofmarrow-derived CD34-/CD38primitive progenitors in methylcellulose, we observed that
MS-S stromal cell linetothe
Day 20
Fig 4. Analysis of the cell compositionof individualclones derived
from CD34+ CD38~ow at days 11 to 13 of culture and at day 2o of
cultures. The results presented are those of five independent experiments that have been pooled representing the analysis by flow cytometry of 93 and 91 clones,respectively. Culture conditions were
serum-free plus a combination of SCF, IL-3, Epo, GM-CSF, G-CSF, IL6, and MGDF in two experiments, and in three others AP plus a
combination ofSCF. IL-3,- EDO.
.. E;. [D)
. . GM-CSF,G-CSF. and I L - ~ (A)
GM; [.).€/GM.
day 7
and R-PE anti-GPA. The second half was labeled with R-PE antiCD14 and FlTC anti-CD15 or FlTC anti-CD1 lb. Cells were analyzed on a FACS sort.
Ulrrustnrcrrrrul srrrdies. Cultured cells were studied by electron
microscopy (EM). They were washed twice
in Hank's medium at
4"C,fixedwith 1.2S% glutaraldehyde in Gey's buffer for IO minutes." washed, and incubated in diaminobenzidinemedium.'" Cells
were then post-fixed with osmium tetroxide. dehydrated.and embedded in epon.Thinsectionswereexamined
with a Philips CM IO
electron microscope (Philips. Eindoven, Netherlands) after lead citrate staining.
Day 13
day 12
38+ 38f 38-
day 18
assay synergized with added human cytokines (see below)
to promote the development of primitive progenitors." Of
note. we observed that at early time points (day 12) during
the culture a fraction of small (<l00 cells) hemoglobinized
colonies containedMK (Fig 1 A). The erythroid compartment
of these colonies was similar in size and time required for
maturation to that of mature BFU-E-derived colonies.3"
Some typical colonies were studiedby morphology and only
contained E and MK cells. These progenitors were easily
BFU-E both by the size of the colonies generated and by
the time (> 18 to 20 days) required for their differentiation.
were also identifiedinplasma-clot
of MK in these mixed
colonieswas confirmed by immunolabeling withan antiCD41 or an anti-CD61MoAb. Usually 2 to 30 MKwere
counted together with 500 to 1.000 erythroblasts. No other
cell type was detected by morphologic criteria. These colonies were not a simple overlapof two types of colonies since
they were still observed at low cell concentration where even
a single colony was grown. This
result was confirmed by
single cell assay (see below). These progenitors were designated BFU-E/MK.
In another series of experiments, we precisely defined the
properties of this progenitor cell with respect to its CD34/
also tried
CD38 phenotype and response to cytokines. We
to define its place in the hierarchy of hematopoietic progenitor cells by investigating if this bilineage association was
purely random or reflects a preferential association between
E and MK
Phenotype of the Entkroin-Megnknnoc?.tic Progenitor
Expression of CD34 and CD38 antigens on the BFU-E/
MK progenitor was doneby plating different subpopulations
of marrow cells in plasma clot and methycellulose assays.
When bone marrow cells were simultaneously labeled with
anti-CD34 and anti-CD38 antibodies. two discrete DoDulations were clearly identified among CD34' cells: cells
brightly stained with CD38 antibodies (referredto as CD38'
Cells). whichrepresent 70% of the CD34'cellpopulation,
CD38 antigen
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Fig 5. Flow cytometry analysis of clonescontaining erythroblasts
and MK. Dot plot analysis oftwo typical BFU-E/MK clones (A and B).
These clonescontained a great majority of GPA' cells and a minority
The CD41'cells have very high forward and side
scatter properties that distinguish them from erythroblasts. More
than 99% of the cells were labeled by both antibodies. CD15 and
CD14 labeling was negative on these two colonies. Two isotypes
controls llgGl FlTC and lgGl PE) are shown, one on a clone negative
for the CD41 marker (C) and the other onan immature erythroid
clone expressing low level of GPA (D).
(CD38'""). This last subpopulation was very heterogeneous
including CD38 negative (CD38-, 10% of the CD34cells) cells that fell within the limits defined by control IgG,
and CD38' cells (20% of the CD34' cells), although the
limit between these two populations was empirical. These
three subpopulations, CD34'/CD38', CD34'/CD38', and
CD34'/CD38-, were sorted and analyzed for their content
inBFU-EIMK progenitors, simultaneously in plasma clot
and methylcellulose assays.
As shown in Table 1 and Fig 2, BFU-EIMK identified in
plasma clot and methylcellulose assays were present in the
CD34'/CD38' or CD34'/CD38- fraction and were not
found in the CD38' fraction. This contrasted with the distribution of mature BFU-E, 90% of which were detected in
the CD38' fraction, an observation that fits with previous
observations showing that expression of CD38 discriminates
between mature (CD38-) and immature (CD38'""') progenitor cells." If one considers as a single category colonies
hemoglobinized at days 10 to 12, a significant proportion of
these (19% to 60%) contained MK, whereas mature BFUE-derived colonies scored in assays initiated with CD38'
cells were exclusively erythroid. There was some variability
in the frequency of the BFU-EIMK-derived colonies among
CD38- and CD38' cells. In some, the BFU-E/MK progenitors were predominantly in the CD38". whereas in others
they were found in the CD38- cell fraction (Fig 2). Although
immature BFU-E (imBFU-E) were enriched in the CD.38and CD38' cell fraction, approximately 40% of them were
contained in the CD38- cell fraction. Therefore, taking into
account only the expression of the CD38 antigen, BFU-E/
MK appears more immature than the imBFU-E. In plasma
clot and methylcellulose, MK progenitors were also enriched
in the CD34' CD38'""' cell population with significant heter-
ogeneity in the size of the colonies (from 3 tomorethan
100 MK). In the CD34' CD38- cell fraction, 30% to 50%
of the MK colonies were bipotent. In contrast, in the CD34'
CD38' cell fraction MK progenitors gave rise to pure MK
Kinetics of development of MK-containingprogenitors
were next analyzed in plasma clot assays. As shown in Fig
3, the kineticsof BFU-WMK and CFU-MK were comparable.
MK colonies were the first colonies to be recognized. and at
day 7 of culture, MK colonies were almost the only type of
hematopoietic colonies present with some colonies containing
up to 50 MK. CFU-MK were counted at dayI2 and contained
up to a few hundred MK. BFU-WMK-derived colonies developed with very similar kinetics: they were first detected at
day 7 of culture and their number was maximum at about day
12. Althoughtheabsolutenumber
of BFU-WMK-derived
colonies was low, these mixedcolonies represented up to 60%
of the total number of pooled MK and E colonies. At day 18
of culture, CFU-MK- andBFU-WMK-derivedcolonies
werestillpresent but begantolyseandwere
not detected
after day 20. In contrast. large E colonies that included granulocytes, monocytes, and more rarelyM K were detected at this
time. By day 18. in methylcellulose cultures, lysingBFUEIMK colonies and growing CFU-GEMM-derived colonies
were observed in the same dish, but they clearly corresponded
to two distinct types of colonies.
Single Cell Cdtlrres
The existence of a bipotent BFU-E/MK progenitor was
clearly established in single cell cultures. Furthermore, this
approach also allowed us to validate previous observations,
which suggests thatevenlate in differentiation the E and
MK programs are preferentially associated. CD34'/CD38'""
cells from five different bone marrows were directly sorted
into wells of 96-well microtiter plates. After sorting, we
checked that a single cell was present in more than 95% of
the wells. Five hundred wells were initiated per experiment
in IMDM supplemented in three experiments with 10% AP
and a combination of six cytokines (SCF, Epo, IL-3, GMCSF, G-CSF, and IL-6) and in the other two in serum-free
medium supplemented with the same cytokines plus MGDF.
This combination of cytokines was chosen to ensure optimal
differentiation in E, MK and granulo/macrophagic pathway.
Cultures were studied after I O to 12, 18 to 20, and in two
experiments after 30 days at 37°C 5% COz. Clones were
selected that contained at least 100 cells and only those were
analyzed. Those represented 8% 2 2% and 20% 1- 6% of the
initial wells at days 12 and 20, respectively. In a preliminary
experiment, individual clones were pipetted off, cytocentrifuged. and morphologically analyzed. However. this procedure resulted in partial loss of MK during cytocentrifugation,
and in subsequent experiments clones were analyzed by flow
cytometry. Each clone was divided into two equal cell fractions. Part of the cells was labeled simultaneously with antibodies against GPA and either CD61 or CD41 and the other
half with antibodies directed against CD14 and either CD15
or CD1 1b. Under these conditions, at day 12. 6156 of the
93 analyzed clones contained some erythroblasts (ranging
from 15% to 100% of the total population) and thus arose
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Table 2. Assessment of Different Progenitor Cell Types in Serum-Free Semi-Solid Clot Assay Supplemented
With Different Combinations of Cytokines
Progenitor Cells per 1,000 CD34'/CD38"
32 2 2
t 1.5
2 3
2 3.7
2 0.57
2 3.4
16.3 i 0.57
20 -c 3.6
11.3 2 2.5
t 4.5
2 2
17.6 t 3
SCF, Epo
12 Epo, SCF
EPO, IL-3,
36.3 C 5
3.6 2 0.57
29.3 -t 16
1 2 1
37.6 t
15.3 z 0.57
25 t 6.5
2 2.5
t 1.5
12.3 i- 0.57
22.3 t 5
2 3
t 1.5
2 0.57
2 0
2 4.9
2 2
11.3 ? 3.7
2 6.6
13 t 4
20 2 3
0.66 2 0.57
2.3 2 1.1
2 1.5
6 5 1
12 2 1.1
7 2 3
2 1.5
5 2 1.7
14 4.5
2 0.57
0.6 2 0.57
CD34+/CD38'OWcells (ie, CD38- CD3V cells)(1.000 per plate) were platedin serum-free semi-solid medium with the indicated combinations
of cytokines. Colonies were scored at days
10 t o 12 after immunolabeling of the plates with anti-CD61 antibody as described
(see Materials and
Methods). Numbers represent the mean c SD of counts performed in three different plates. Results are from one experiment.
from a progenitor capable of erythroid differentiation (Fig
4). Twenty-one percent of the clones (pooled experiments)
contained only GPA+ cells, while 30% of the clones contained both GPA' cells and cells highly labeled with the
antibodies against the CD61 or CD41 antigen (Fig 5). These
cells positive for MK markers had high forward and side
scatter properties characteristic of cultured MK, which allow
easy discrimination between erythroblasts and MK on the
basis of light scatter properties. In all these clones, GPA+
cells represent more than 85% and MK 1% to 8% of the
events analyzed (Fig 5). None of the cells present in these
clones expressed granulocytic markers (CD14 and CD15).
Clones containing granulocytes andMK without erythroblasts or granulocytes and erythroblasts without MK were
also observed but at a low frequency (3% and 4%, respectively). Since we selected only wells containing over 100
cells, pure small MK clones could not be detected by flow
cytometry. However, these were detected by microscopic
observation and their MK nature was further documented by
CD41 immunolabeling (data not shown).
At day 18 to 20 of culture, 20% of the total number of
wells contained more than 100 cells and 56% of the 91
analyzed clones contained cells that expressed either CD14
or CD15 or both. A minority of the clones (8%) only contained GPA' cells. BFU-E/MK clones were also uncommon
(5%) withveryfewMK
(<2%). In contrast, clones containing GPA+, CD14+, CD15+,and CD41+ cells were much
more frequent (22%) suggesting that they were derived from
CFU-GEMM. These clones contained on average 5-fold
more cells than those studied atday 12. Later in culture
(around day 30), all the clones analyzed contained CD14'
and CD15' cells but no GPA' or CD41+ cells.
Two BFU-E/MK clones identified at day 12 were subsequently studied at day 18, and no granulocytic cells could
be detected.
Dejnition of Optimal Conditions for the Detection of
BFU-E/MK Progenitors
The BFU-E/MK requirement for cytokines was first assessed in plasma clot cultures, which are optimized for the
expression of MK differentiation. Cultures were ended at
days 10 to 12 and stained withan anti-CD61 MoAb as
Table 3. Effect of the Addition of MS-5 Cells on the Growth of Different Types of Progenitor Cells in MethylcelluloseAssays
Progenitor Cells per 1,000 CD34+/CD38'"" Cells
+ IL-3 + EPO
34 2 17
27 5 7
18 2 7
34 t 17
21 2 4
18 7
6 2 1
23 2 7
18 2 5.5
5 2 1.5
3 +3
20 2
31 -c
13 2 5
2 1.7
20 2 4.5
7 2 2
11 2 2.5
One thousand CD34'/CD38'OW (ie, CD38CD38' cells) were plated in methycellulose colony assays in the presence of either A-LCM + Epo
cells per dish.E/MK were enumerated
or SCF IL-3 + Epo as growth factors and30% FCS. MS-5 cells were added at the concentration10.000
in the mBFU-E and scored at day 12 and CFU-GEMM in the imBFU-E and scored at days 20
t o 25. Duplicate plates were initiated in each
condition. Numbers refer to the mean t SEM of counts obtained in the indicated number of experiments (n).
From by guest on December 3, 2014. For personal use only.
Fig 6.
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Fig 6. Ultrastructural studies of an MK cultured from CD34' CD38- cells in liquid culture in the presence of MS-5 cells, a combination of
SCF, IL-3, and AP. CD34' CD38" cells were grown in liquid culture in the presence of a confluent layer of MS-5. At day 9, a nearly pure MK
population was present, whereas later in cultures (day 14 andafter1 other cell types were predominant. (AI A rosette with a large MK
surrounded bytwo MS-5 cells. Note thatMK develops blebsin the cdntact with MS-5 cells. Magnification x 2,900. (6)Enlargementof the MK
cytoplasm. There isa harmonious distribution of demarcationmembranesand a-granules. Magnification x 19,600. (ClZone of contact between
MK and an MS-5 cell. MS-5 cells posess numerous large granules and cisternae of endoplasmic reticulum. The cell membrane extends its
pseudopods between thelarge blebs of MK,which are always devoid of organelles. Note the tight contact between the twocells. Magnification
x 7,700.
described.32In a first set of experiments performed before
the cloning of MGDF (MpI-L, TPO), we compared the effect
of normal human and aplastic human plasma on the development of CD34+/CD38low (including both CD38- and
CD38'). In the presence of normal plasma, the growth of
BFU-E/MK and CFU-MK was optimum in the presence of
the combination of SCF, IL-3, and Epo with 4 ? 1.4 BFUE N K per 1,000 cells, and 6 5 2.8 CFU-MK per 1,000 cells.
Similar results were found with AP, but cloning efficiency
was slightly higher ( 5 ? 1.4 BFU-E/MK per 1,000 cells,
and 9.5 2 1.9 CFU-MK per 1,000 cells). In a second set of
experiments, we tested the effect of MGDF in serum free
conditions (Table 2). Best results were obtained when
MGDF was added to SCF, IL-3, Epo with 13 to 17 BFU-E/
MK per 1,000 CD34+/CD3g1""(including CD38- + CD38')
cells, and 25 to 27 CFU-MK, whereas only six BFU-E/MK
progenitors were observed without MGDF. Withdrawal of
either IL-3 or SCF even though high concentrations of
MGDF were kept constant decreased the number of BFUE/MK progenitors, but not of CFU-MK, which fits well with
the known requirement of either IL-3 or SCF for the proliferation of early E cells. Addition of IL-6 either to a combination of three (SCF, IL-3, Epo) or four (the same three +
MGDF) did not further increase plating efficiency. Precise
quantitation of the E compartment was hampered in plasma
clots by the early termination required for MK quantification.
Methylcellulose assays were also performed with CD34'
CD38'"" cells in the presence of 30% FCS, a concentration
that is optimum for E development but selectively inhibits
MK d e ~ e l o p m e n t ? Addition
of A-LCM or the combination of SCF, IL-3, and Epo and even MGDF did not neutralize this inhibition. In contrast, the addition of MS-5 cells
tothe assay (independently of the cytokine combination)
dramatically counteracted FCS effect both on CFU-MK and
BFU-E/MK (Table 3 and Fig IC). Thus, on average eight
CFU-MK were scored per 1,000 CD34+/CD38'"" cells (Table l). Most interestingly, even though the total number of
small E colonies scored as mBFU-E in the CD38loWfraction
did not change when MS-5 cells were added (Table 3), 20%
to 25% of these now clearly included MK, which suggests
that a proportion of E progenitors were in fact not restricted
to the erythroid lineage. The observation that 90% of the
mBFU-E are in the CD38+ fraction and generate only E cells
independently of the presence of MS-5 cells reinforced the
idea that theBFU-E/MK progenitor represents a unique step
in the hierarchy of progenitors. However, expression of the
MK potential requires specific conditions, and in the absence
of MS-5 cells, this progenitor would be scored as a mature
BFU-E only capable of differentiating along the E pathway.
Of note is the fact that MS-5 cells also increased the size of
MKS suggesting thattheymay
also act on MK terminal
differentiation (see EM studies below).
In contrast to their lack of proliferative effect on small E
colonies, the number andsize of which did notchange (Table
1 and ref 26), MS-5 cells stimulated the proliferation of
immature BFU-E and CFU-GEMM as 20 to 30 of these
progenitors were detected per 1,000 CD34+ CD38- cells,
but almost none in the absence of MS-5 cells (Table 3).
Similar results were observed in plasma clot, after immunolabeling. BFU-E/MK and pure MK colonies were absent
when cultures contained 30% FCS. MK differentiation was
observed when MS-5 cells were added.
Ultrastructural studies confirmed the action of MS-5 cells
in promoting terminal MK maturation. For these studies,
purified CD34+/CD38Io"cells were cultured for 7 to 10 days
in liquid medium in the presence of MS-5 cells plus AP with
or without SCF and IL-3 and studied by EM. These cultures
were compared with control cultures performed in the absence of MS-5. In 10 distinct experiments, MK differentiation occured in close contact with MS-5 (Fig 6). MK were
generally surrounded by MS-5 in a rosette-like shape. In the
areas of contact with MS-5. MK developed numerous blebs
that were absent outside this zone. Contacts between the two
membranes wereverytightbutwithout
junction. In some
rosettes, MS-5 cells extended their pseudopods between each
bleb. The quality of maturation was equivalent to that observed in vivo with the regular distribution of demarcation
membranes and a-granules. No such degree of maturity was
observed in control cultures (without MS-5). Adhesion between MK and MS-5 cells occurred very early during maturation since small promegakaryoblasts with large nucleoli
were associated with MS-S cells. No such intimate contacts
were seenwith cells from other lineages, granulocyte or
Cell Cycle Status of the BFU-E/MK Progenitor
Results from the experiments described above suggest
that BFU-E/MK progenitors are more immature than the
CD38+ E-restricted progenitors based on their lack of expression of CD38 and ability to differentiate into two lineages. A third indicator of primitiveness is quiescence.6 In
this series of experiments, we determined the cell cycle
status of BFU-E/MK by three-color labeling using R-PE
anti-CD34 MoAb, FITC anti-CD38 MoAb, and Hoechst
dye as shown in Fig 7A and B. In an attempt to increase
the sensitivity of the procedure and detect subpopulations
within the GO/GI population, which may correspond to
more primitive populations, incubation with the Hoechst
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C M 8 FlTc
Fig 8. Cell cycle status of BFU-EIMK progenitors. CD34'CD38'""
cells were subfractionated according t o the Hoechst staining in a
Hoechst'"", GOIGl, and SIGZIM cell subfractions as shown in Fig
6. Cells were grown in fibrin clot in serum-free conditions using a
combination of SCF, 11-3, Epo, and MGDF. Results are the number of
colonies per 1,000 plated cells and are the average of one experiment
made in triplicate. Two other experiments gave similar results.
1O M )
Fig 7. Hoechst 3342 staining of CD34'cells.CD34'
cells were
purified by the immunornagnetic
bead technique (Dynal) and subsequentlylabeledby
R-PE-anti-CD34, an FITC-anti-CD38, andthe
Hoechst dye..(A) A dot plot analysis between the CD34 and CD38
labeling isshown. Only the CD34h"8hcells were analyzed for Hoechst
staining and sorted into
t w o cell fractions, CD38* and CD38'"". (B) A
dot blotanalysis between theCD38 staining and the
Hoechst labeling
in the CD34highgate is shown. Three populations as shown in the
histogram (C) can be observed according to the Hoechst staining:
Hoechst'"", GOIG1, and SIGZIM.Of note, cycling cells are essentially
in the CD38' cell fractions, whereas Hoechst'"" cells are in theCD38'""
cell fraction.
stain was shortened, whereas usually 2 hours is required
to reach the equilibrum. With this procedure, three CD34'
populations were identified, Hoechst', which may identify more primitive progenitors,'5 Hoechst" (2n), and
Hoechst" (S/G2/M) (Fig 7, B and C). There was a clear
correlation between the expression of CD38 and the uptake
of the Hoechst dye (Fig 7B). Among the CD38'""' subset,
all cells were Hoechst' or Hoechst' and only 1% to 4%
of the cells were in the Hoechst" (S/G2/M) fraction. In
contrast, 10% to 15% of the CD38' cells was in the S/G2/
M phase. Since a similarly low proportion of CD38'"" cells
was in the S/G2/M phase when the Hoechst staining period
was prolonged for 2 hours, the number of cycling cells
was not underestimated. In addition, this result is in
agreement with a recent report using propidium iodide to
stain DNA." Cell sorting experiments were held in which
CD34' CD38- Hoechst'"", CD34' CD38- Hoechst' (GO/
GI), and CD34' CD38- Hoechst" (S/G2/M) cells were
assayed for their contentin progenitors. The results showed
that most BFU-E/MK progenitors were in the Hoechst' or
Hoechst' fractions and only 0.32% and 0.95% of BFU-E/
MK and CFU-MK of the CD38'""' population were in the
S/G2/M phase of the cell cycle, respectively. Cloning efficiency of BFU-E/MK was much higher in the CD34'
CD38- GO/GI cell population than in the two other cell
fractions (1.2% versus 0.28% and 0.25%) (Fig 8). Further
functional analysis of these different Hoechst fractions for
their content of other clonogenic progenitors of varying
maturity showed that 20% of the mBFU-E contained in
the CD38' cell fraction were in the S/G2/M phase of the
cell cycle. In contrast, only 1.2% of the mBFU-E and less
than 0.5% of the CFU-GEMM contained in the CD38- cell
fraction were in S phase.
Observations of mixed' MK-E bursts in mice" and also
in humans'"have provided the initial basis suggesting the
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existence of a bipotent progenitor restricted to both MK and
E lineages. Further studies have suggested that divergence
of both lineages was a late event in the hierarchy of stem
cell differentiation4 although no direct evidence supported
this hypothesis. Since then, considerable evidence in support
of such a common pathway has been obtained from studies
that showed that several transcription factors such as GATA1, Tal- 1 , and NFE-2 are shared by both lineages. l4,l6 Detailed
data on the phenotype and growth properties of this bipotent
progenitor are lacking partly because most of the studies
were performed before the isolation of the different hematopoietic growth factors and the development of cell purification techniques. Since then further similarities in the response to Epo and MGDF of committed progenitors from
both lineages and shared expression of Epo-R also strengthened the hypothesis. In this study we provide strong evidence
that a bipotent MK-E burst exists in human marrow, which
can be distinguished from its more mature progeny, CFUMK and mature BFU-E and also from its immature ancestor
CFU-GEMM, by its phenotype, cell-cycle status, and cytokine response.
If one assumes that colony size and time taken togenerate
erythroblasts and mature MK both reflect the state of differentiation of the progenitor, BFU-E/MK appeared to becloser
to mature BFU-E than to immature BFU-E or CFU-GEMM,
based on the fact that mixed colonies were small containing
at most 1,000 erythroblasts and a variable number ofMK
from 2 to 30."' The number of BFU-E/MK was maximal
after a period of 12 days in culture and colonies lysed a few
days later. In contrast, 16 to 18 days must elapse before
primitive BFU-E and CFU-GEMM can express their full
potential and complete differentiation. If the kinetics of colony formation classifies BFU-E/MK as close to mBFU-E,
they differ from these both by their phenotype and cell cycle
status. In contrast to day 12 BFU-E, 90% of which express
both CD34 and CD38 antigens, BFU-E/MKwere in the
CD34' CD38'"" (either CD38' or CD38-) fraction and the
great majority ( S O % ) of BFU-E/MK were in GO/G1 phase
of the cell cycle, two properties thatusually characterize
immature progenitors.".." Noteworthy, this was also true for
a fraction of CFU-MK, therefore suggesting that CD38 may
not be expressed by cells of the megakaryocytic lineage to
the same extent as on other myeloid precursors.
Definite proof that the potential of BFU-E/MK was restricted to E and MK lineages only was obtained in single
cell experiments. The frequency of BFU-E/MK was estimated from the proportion of wells that contained more than
100 cells and only GPA' and CD41' cells after 12 days in
culture. An estimated 2% to 3% of total wells initiated with
CD34' CD38' contained BFU-E/MK based onthese criteria,
which represented 0.3% to 1% of normal marrow CD34'
cells. By comparison, the frequency of clones containing at
least three lineages and thought to originate from standard
CFU-GEMM was 4.4% in the CD34' CD38' and 1% in the
initial CD34' cell fraction. Despite the fact that these numbers might be underestimated, as only clones containing
>l00 cells were analyzed by flow cytometry at day 12, they
were in agreement with frequencies of BFU-E/MK calculated from colony assays. Finally, it remains to be determined
whether some lineage markers (especially CD41 as suggested by a recent report15) could be expressed on E/MK
progenitors. Our preliminary results do not favor this hypothesis as the CD34'CD41'cell
population was essentially
enriched in MK progenitor^.^^
In vitro appraisal of the real potential of progenitors is
hampered by two difficulties. The first pertains to the fact
that specific culture conditions and cytokine combinations
that optimally support the differentiation of progenitor cells
committed to one lineage may be inhibitory for progenitor
cells committed to other lineages. Thus, myeloidand B
lymphoid differentiations in long-term cultures require conditions that are mutually exclusive, but this is also true in
colony assays where IL-3 has recently been shown to inhibit
the emergence of B220+ cells in colonies." In this study,
bipotent E/MK progenitors were notdetected in the standard
conditions of methylcellulose colony assays optimized for
the growth of primitive BFU-E, ie, 30% FCS plus SCF, IL3, and Epo. In the presence of FCS, the addition of MS-5
stromal cell line was absolutely required for the growth of
E/MK progenitor and CFU-MK and no combination of cytokines could replace these stromal cells. In contrast, when
normal plasma or AP were substituted for FCS, MS-5 cells
improved terminal MK differentiation but had no significant
effect on the number of MK andE/MK colonies. The inhibitory activity present in serum that inhibits MK gr~wth",".~'
is most likely accounted for by TGF-p.23,40.4'
Two potential
mechanisms, which might act in concert, may be hypothesized to explain the effect of MS-5 cells: (1) adsorption of
inhibitory molecules on proteoglycans synthesized by stromal cells may result in their neutralization, a mechanism
well documented for TGF-p42;(2) alternatively, stromal cells
may secrete cytokines able to counteract this suppressive
effect as recently reported for bFGF.43Whether this is accounted for by MGDF is as yet unclear; recent experiments
has reported for other stromal
cells,@ synthesizes low amounts of MGDF (F. Wendling,
manuscript in preparation). However, exogenous MGDF
added to colony assays with 30% FCSin the absence of
MS-S supported the growth neither of BFU-ENK nor CFUMK. In serum-free plasma clot assays, MGDF alone optimally recruited CFU-MK but could not replace SCF or IL3 in the E/MK progenitor assay. This, together withthe
observation that addition of MGDF to the combination of
SCF, IL-3, and Epo increased the cloning efficiency of E/
MK progenitors suggests that optimal development of E/MK
progenitors requires combinations of cytokines that probably
act sequentially on progenitors at different maturation stages.
Further experiments will be required to characterize MS-Sderived molecules that affect MK maturation.
Two additional parameters may lead to underestimation
of the real potential of progenitors in culture: asynchrony in
the acquisition of specific differentiation markers identifying
commitment to different lineages during the course of clone
formation and discrepancy in the number of cells representative of each lineage.5 For example, emergence of lymphoid
cells in colonies grown from iymphohematopoietic progenitor inthe mouse is a very late event.45Three arguments,
however, rule out that E M K clones were in factmultilineage
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clones: (1) a similar phenotype was observed in clones that
were sequentially studied at days 12 and 18; (2) time course
studies in methylcellulose colony assays indicated thatE/MK
and CFU-GEMM represent two successive nonoverlapping
waves of progenitor cells; (3) the sensitivity of immunolabeling of individual colonies, which allows the recognition of
one to two MK cells within a colony.
Recent advancesin the characterization of molecules associated with commitment to E and MK differentiation also
strengthen the hypothesis of atightlinkage between both
lineages. Expression of several transcription factors such as
GATA-I, Tal-l, and NFE-2 is primarily found in both lineages,l?,14,16.17,4~~U
and at least in vitro, GATA-I plays a pivotal role for expressionof both E and MK genes.“ However.
recent experiments suggesting that GATA-1 is only required
during the late stages of the E differentiation4’ and the lack
of a majordefect inmegakaryocytopoiesis
knock-out mice,’” question the real requirement of GATA1 expression forearly E and MK development.Whether
GATA-I has aninductive role in the commitmentof pluripotent stem cells as recently suggested in an avian cell line”
or if its lineage-specific effects may be reiated to its restricted
question. Our
pattern of e ~ p r e s s i o n ”is~still
~ ~ an
~ ~unsolved
results suggest that in the near future, it may be possible to
have access to cellular models that directly allow us t o address these questions in specific cell subpopulations.
Such an assay, which allows simultaneous identification
of a high number of bipotent progenitors as well as their
monopotentprogeny, will alsobe useful to characterize
growth requirements of each compartment, with particular
emphasis on the response to Epo and MGDF. The Eand
MK lineages are the only two hematopoietic cell lineages
that are regulated by humoral factors (Epo and Mpl-L/TPO/
MGDF), the level of which is regulated by the mature cell
mas^.'^-^'' Several lines of evidence suggest that the Epo-R
is expressed on M K ” and conversely it cannot be excluded
on asubset of E progenitors.”
However, preliminary attempts to identify Epo-R and MplR on BFU-EMK progenitors by flow cytometry have failed
(data not shown), but this does not exclude the possibility
that E M K progenitors express both the Epo-R and Mpl-R
at very low, albeit biologically active, levels.
After acceptance of this report, a report
also demonstrating
the existence of bipotent E and NK progenitors was published.“
We thank S. Gillis (Immunex, Seattle,WA) for a gift of IL-3, IL6 , and GM-CSF and P. Hunt (Amgen, Thousand Oaks, CA) for the
gift of megakaryocyte growth and development factor.We are grateful to F. Lepesteur and B. lzac for technical assistance. The Y2/51
MoAb was a gift from Dr D. Mason (Oxford, UK). We are grateful
to the surgeons for providing the bone marrow samples. We also
thank S. Burstein for improving the English manuscript.
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