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RAPID COMMUNICATION
Expression of the YB5.BS Antigen (c-kit proto-oncogene product)
in Normal Human Bone Marrow
By Leonie K. Ashman, Antony C. Cambareri, L. Bik To, Roland J. Levinsky, and Christopher A. Juttner
The c-kit proto-oncogene product is a member of the family
of growth factor receptors with intrinsic tyrosine kinase
activity. In the mouse c-kit maps t o the W locus, which is
known to be of central importance in hematopoiesis. Monoclonal antibody (MoAb) YB5.B8, which was raised against
peripheral blood blast cells from a patient with acute myeloid
leukemia (AML), was recently shown t o bind t o the extracellular domain of the c-kit product. This antibody does not bind
detectably t o normal peripheral blood cells and identifies a
sub-group of AML patients with poor prognosis. We have
used MoAb YB5.B8 t o study the expression of c-kit by normal
human bone marrow cells by immunofluorescence and flow
cytometry, and t o isolate multipotential and erythroid colonyforming cells. In a series of 11 normal adult bone marrow
specimens, MoAb YB5.B8 bound to 4.0% k 1.8% of the cells
in the low-density fraction. Dual-labeling experiments were
perfarmed with YB5.B8, and CD33, CD34, and CDlO MoAbs.
Three populations of cells binding YB5.B8 could be identified
based on their pattern of coexpression of the other markers;
ie, YB5.B8+/CD34+/CD33-, YB5.08'/CD34+/CD33'
and
YB5.B8'/CD34-/CD33+. These populations had distinctive
two-dimensional light scatter characteristics and are likely t o
correspond t o precursor colony-forming cells, colony-forming cells, and maturing mast cells, respectively. No cells
binding both YB5.B8 and an MoAb to the early lymphoid
marker CDlO were found, implying that most early lymphoid
cells do not express c-kit. MoAbs to the c-kit protein should
prove valuable in multimarker studies of human hematopoiet i c stem and progenitor cells. Definition of a reference range
of c-kit expression in normal human bone marrow will
provide a sound basis for further studies of this marker in
diagnosis and prognosis in AML.
0 1991by The American Society of Hematology.
T
Recently we showed that a murine monoclonal antibody
(MoAb), YB5.B8,I4 raised against blast cells from a patient
with acute myeloid leukemia (AML) of M1 sub-type binds
to the human c-kit proto-oncogene product." This MoAb,
which fails to bind to any cells in normal peripheral blood,14
was known to bind to a low copy-number antigen expressed
by a small proportion of normal bone marrow mononuclear
cells, including hematopoietic progenitors that give rise to
colonies of neutrophils, macrophages, and eosinophils in
vitro.I6 Furthermore, inclusion of the antibody in the assay
of colony-forming units in culture (CFU-C) resulted in
inhibition of factor-dependent colony
suggesting
that YB5.B8 binds to a growth factor receptor. However,
the antibody did not bind to the receptors for any of the
known growth factors for myeloid cells, ie, granulocytemacrophage CSF (GM-CSF)," interleukin-3 (IL-3), IL-4,
CSF-1, or granulocyte CSF (G-CSF) (L.K. Ashman, unpublished data, 1989, 1990). The similarities between the
YB5.B8 antigen and the c-kit product, notably their similar
molecular weights (145 Kd)24L8
and their similar very
restricted cellular distribution, especially the strong expression on mast cells,"~''~"suggested that the two might be the
same. This suggestion was confirmed by sequential immunoprecipitation experiments using MoAb YB5.B8 and a
polyclonal antiserum to v-kit that binds to the cytoplasmic
domain of the c-kit product.''
Because MoAb YB5.B8 binds to the extracellular domain of the c-kif receptor in intact cells, it provides a means
of determining the pattern of c-kit expression in normal
human bone marrow populations, and of purifying c-kit
positive cells for functional studies. In this communication
we describe analytical flow cytometric studies of the pattern
of coexpression of the c-kit product and other progenitor
cell markers, namely the CD10, CD33, and CD34 antigens,
by normal bone marrow cell populations. In addition, we
have extended our earlier studies to show that erythroid
and mixed myeloid progenitor cells (CFU-mix), as well as
HE KIT ONCOGENE was initially identified as the
transforming element of the HZ4-feline sarcoma virus.' Subsequently the normal cellular homolog, c-kit, was
identified in human2 and m u ~ i n ecells,
~ . ~ and was shown to
encode a receptor tyrosine kinase with strong homology to
the receptors for colony-stimulating factor 1 (CSF-1) and
platelet-derived growth factor. Recently the ligand for c-kit
was identified' '
The c-kit proto-oncogene is known to play a key role in
murine hematopoiesis; c-kit maps to the white spotting ( W )
locus9~"and the kit ligand (KL) is the product of the Steel
(9)~ O C U S . ~Mutations
~'~~~
at these two loci produce a very
similar phenotype characterized by severe anaemia and
mast cell deficiency. Melanocyte development and gametogenesis are also affected. The defective hematopoiesis in W
mutant mice can be corrected by transplqntation of bone
marrow stem cells from normal syngeneic mice, whereas the
SI mutation appears to affect stromal cell f~ncti0n.l~
It
seems likely that c-kit is also important in human hematopoiesis.
From the Department of Microbiology and Immunology, The
University of Adelaide; the Division of Haematology, Institute of
Medical and Veterinary Science, Adelaide, South Australia; and the
Hugh Greenwood Department of immunology, Institute of Child
Health, London, UK.
Submitted February 14, 1991; acceptedApril 11,1991.
Supported by grants to L.KA. from the Rotary Peter Nelson
Leukaemia Research Fund and the National Health and Medical
Research Council OfAustralia.
Address reprint requests to L .K. Ashman, PhD, Department of
Microbiology and Immunology, The University of Adelaide, G.P.0.
Box 498, Adelaide, SA 5001, Australia.
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.
0006-4971/91/7801-0036$3.00/0
30
Blood, Vol78, No 1 (July 1). 1991: pp 30-37
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31
C-KIT EXPRESSION IN NORMAL BONE MARROW
CFU-GM, express c-kit, and have defined a reference range
for c-kit-positive cells in normal bone marrow.
MATERIALS AND METHODS
MoAbs. MoAbs YB5.B8 (IgGl)I4 and the negative control
IgGl MoAb Sal-2” (anti-Salmonella) were purified from culture
supernatants by affinity chromatography on Protein A-Sepharose2’
(Pharmacia-LKB, Piscataway, NJ). For use in progenitor cell
isolation, aliquots of purified antibody (200 pg/mL) were extensively dialyzed against phosphate-buffered saline (PBS) followed
by culture medium (RPMI-1640) to remove azide. Fetal bovine
serum (FBS) was added to a final concentration of 10% volhrol, and
the antibodies sterilized by filtration through a 0.22-c~mmembrane
(Millex GV; Millipore, Bedford, MA), then diluted to 20 Fg/mL in
medium/lO% FBS. CD34 MoAbs ICH-3 (IgG2a)22and BI-3C5
(IgG1)z2.23
were used as 1/500 dilutions of ascites for immunofluorescence and, where applicable, for cell isolation following dialysis
as described above. The CDlO IgG2a MoAb J524(Coulter, Hialeah,
FL) and the CD33 Ig2b MoAb M P z 5 (Coulter) were diluted
according to the manufacturer’s instructions and used for immunofluorescence experiments only. Negative control IgG2a and
IgG2b MoAhs, Sal-5 and Sal-1,” respectively, were used as
undiluted culture supernatants. Positive control antibodies were
anti-HLA class I MoAb 1B4 (IgG1) and 7B6c (IgG2a)26prepared
in this laboratory and used as culture supernatants.
Bone marrow cells. Normal bone marrow aspirates were obtained with informed consent and with the approval of the Ethics
Committees of the Royal Adelaide Hospital and the University of
Adelaide from normal adult volunteers, from bone marrow donors
participating in the allogeneic transplantation program, and from
patients with nonhematologic malignancies undergoing bone marrow biopsy for clinical purposes. The low-density fraction was
isolated from the aspirate by centrifugation on Ficoll-Paque
(Pharmacia-LKB) and the cells isolated from the interface were
washed three times in medium 10% FBS.
Immunofluorescence assay. Normal bone marrow mononuclear
cells were washed three times in PBS-0.1% bovine serum albumin
(BSA)-0.1% sodium azide at 4°C and resuspended at 107/mLin the
same buffer containing 5% normal rabbit serum and 5% normal
human serum (both heat-inactivated) to block Fc receptormediated binding of MoAbs?? Aliquots of 50 bL were incubated
with 50 IJ.Lof MoAb, diluted as indicated, at 4°C for 45 minutes,
then washed three times as above. For single-color immunofluorescence cells were then incubated far a further 45 minutes at 4°C with
fluorescein-labeled affinity-purifiedF(ab‘)zsheep antibody to mouse
Igs (Silenus Laboratories, Hawthorn, Australia, code DDAF).
After a further two washes, cells were fixed with 1% paraformaldehyde in PBS and stored at 4°C in the dark for up to 3 days before
analysis on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
For two-color immunofluorescence, isotype-specific secondstage reagents were used. IgGl MoAbs were detected with
R-Phycoerythrin (PE)-labeled goat antibody to mouse IgGl (Caltag,
San Francisco, CA). IgG2a and IgG2b MoAbs were detected with
fluorescein-conjugated goat antibodies to mouse IgG2a and IgG2b
(Caltag), respectively. Second-stage incubation was for 45 minutes
at 4°C followed by two washes, as above. After labeling, cells were
fixed and aualyzed as above, except that cells in the neutrophil
region of the two-dimensional scatter plot, which were uniformly
negative with both YBS.B8 and CD34 MoAbs, were excluded from
the analysis. Color compensation was set using cells labeled with
MoAbs to HLA Class I antigens as the first stage. Percentages of
positive cells in the different regions were determined using
Paint-a-Gate software (Becton Dickinson) and corrected for the
+
negative control (isotype-matched controls in place of first-stage
MoAbs).
CFU-mix assay. Freshly isolated low-density bone marrow cells
were depleted of monocytes by adherence to plastic for 1 hour at
37T, then separated into “positive” and “negative” fractions by
immune rosetting using MoAbs YB5.B8, BI-3C5, and Sal-2 (all
IgGl isotype) essentially as described previously.16 Briefly, cells
were suspended at 107/mLin medium/lO% FBS and incubated for
1 hour at 4°C with an equal volume of dialyzed MoAb. Following
two washes with medium/lO% FBS, cells were resuspended to the
original volume and incubated with an equal volume of human
erythrocytes coated with affinity-purified goat antibody to mouse
Igs (prepared in this laboratory) by the chromic chloride method.
Cells were pelleted by centrifugation at 2 w , followed by incubation at 4°C for 60 minutes. After gentle resuspension, cells were
layered onto Ficoll-Paque (Pharmacia) and centrifuged at 400g for
25 minutes. Unrosetted cells were recovered from the interface
and rosetted cells were recovered from the pellet. Cells from both
fractions were washed three times with medium/lO% FBS at room
temperature before plating as described below. Where sufficient
material was available, additional cells were rosetted to enable the
yield of cells in the two fractions to be determined. The yield
totalled approximately 50% of the starting number.
The CFU-mix assay was performed as previously described.*”’
Cells fractionated by the immune rosetting technique were cultured in 0.9% methylcellulose (Fluka, Buchs, Switzerland) with
Iscove’s modified Dulbecco’s medium (Commonwealth Serum
Laboratories, Parkville, Australia), 30% volivol FBS (GIBCO
BRL, Glen Waverley, Australia), 0.66% deionized BSA (fraction
V, Sigma, St Louis, MO), 20 p”l/L 2-mercaptoethanol, 5%
vol/vol phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), and 1UimLpurified human urinary erythropoietin (Toyobo, Osaka, Japan). Cultures were incubated in a humidified atmosphere of 5% CO, in air at 37°C for 14 days. Twenty to 25
replicate plates were used for each fraction; each plate received the
rosetted or unrosetted cells derived from 1 x lo5 low-density
nonadhereqt bone marrow cells.
Large ( > 100 cells) colonies with a central hemoglobinized area
surrounded by nonhemoglobinized cells were scored as CFU-mix.
Smaller (approximately 40 to 100 cells) densely hemoglobinized
colonies were scored as burst-forming units, erythroid (BFU-e) or
colony-forming units, erythroid (CFU-e), respectively, depending
on whether they were fragmented or compact. Nonhemaglobinized
colonies (> 40 cells) were classified as “myeloid” (CFU-c).
RESULTS
DeJinition of a reference range for cells expressing c-kit in
normal bone marrow. Low-density bone marrow cells from
11 hematologically normal adult donors (seven diseasefree; four with diseases not involving the bone marrow)
were screened by single-color immunofluorescence for
binding of MoAbs YB5.B8 (anti-c-kit), ICH-3 (CD34), and
isotype-matched negative control MoAbs Sal-2 and Sal-5.
Relative t o the negative control MoAbs, a mean of 4.0%
(SD 1.8; range 1.4 to 6.5) of the cells expressed c-kit
compared with a mean of 2.2% (SD 1.6; range 0.6 to 5.6)
that were positive for the CD34 antigen. The proportion of
cells expressing the CD34 antigen is similar to that (1%to
4%) reported by other^.^^^^*^' As previously shown,I6 the
intensity of MoAb YB5.B8 binding to positive cells was only
about half of that observed with the CD34 l r i < i b (not
shown). When only data from the seven diseaseti -e donors
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ASHMAN ET AL
32
Thc pattern of cocxprcssion of c-kit protein detected by
MoAb YB5.B8 and the CD34 antigen detected by MoAb
ICH-3 was cxamincd on eight normal bone marrow specimens. An example is shown in Fig I , which clearly demonstrates that thc two antibodies bind to overlapping but
nonidentical populations. Furthermore, it can be seen that
the single- and double-positive populations each have
distinctive two-dimensional scatter characteristics (highlighted cvents, Fig I ) . The YB5.B8'/CD34- cells were in
gcncral very largc cells, many of which also had high side
scatter (Fig 1A). These may bc mast cell progcnitors,
although isolation and specific staining is necessary to
confirm this. These cells also stain with My-9 (CD33) (see
below), as do tissue mast cells.'? Cells double-stained with
YB5.BS and the CD34 MoAb fell into the typical blast cell
fraction, which lay just below the monocytes on the twodimensional scatter plot (Fig 1 B). YBS.B8-/CD34* cells
cxtcnded into thc small lymphocyte-likc rcgion as well as
the blast cell region (Fig 1C).
Cocxpression of c-kit with the CD33 antigen detccted by
MoAb My-9 was also examined on specimcns from three
donors. CD33 MoAbs bind to a considerable proportion of
were considered, the mean percentage of cells positive for
YB5.B8 was 4.3 (SD 1.1, range 2.5 to 5.3).
Co-expression of c-kit with other progenitor cell markers.
In earlier singlc-color immunofluorescence experiments we
showed that both the CD34 MoAb BI-3C5 and MoAb
YB5.BS bound to about 25% of the cclls in the blast
fraction (based on two-dimensional light scatter charactcristics) of low-density cells from normal bone marrow.''
Furthermore, both antibodies could bc used to enrich for
hematopoietic progenitors (CFU-c), which gave rise to
colonies of mature myeloid cells in vitro.'' Therefore, it
seemed important to determine whether the two antibodies
bind to identical or overlapping cell populations. Because
neither YB5.B8 nor most available CD34 MoAbs can be
directly labeled without loss of activity, this was undertaken
using isotype-specificsecond-stage reagcnts. The specificity
of these reagents was confirmed in preliminary immunofluorescence experiments using MoAbs directed against HLA
Class I antigens in a criss-cross fashion as the first stage (not
shown). The PE label was chosen for detection of c-kit
protein, bccause this marker appears to be expressed at
lower copy number (see above).
A
:
Q)
m
>-
... - .
....:
,
.. .. .*
. . ..
.
..:
2
v,
9
LL
. . . .<.: .
..
. ..
.
I
'.
.
*
,
I
.
f
. .... ..
.
.
.:
.:%..
. .
)
Fil
- CD34
DL
. .
:.;I:
:.f.:.;,.
I . .
.'.:
.
:,
.
FSC
.,. .,
I
. .
G
FSC
. +
..
. ' -..
.. - .. .. . .,
. *. . * .
.
.*..
..'. .
_. . .
I'
.
Fil - CD34
0'
FSC
Fig 1. Pattern of coaxpression of c-kit and CD34
antigen on normal human bone manow cells. Cells
were labeled with MoAbs YBS.W(IgG1) and ICH3(lgGZa)followed by fluoresceinated goat antimouse
lgG2a and PE-antimouse IgG1. The figure s h o w the
two-color fluorescence dot-plot with the different
regions highlighted (left hand side). On the right hand
side, the two-dimensionallight scatter plots with the
corresponding cells highlighted are depicted. (A)
YB5.B8 single-positivecells; (B) double-positivecells;
(C) ICH-3 single-positive cells.
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33
C-KIT EXPRESSION IN NORMAL BONE MARROW
cclls in normal bonc marrow, including committcd mycloid
progcnitors and cclls of thc monocytic lincagc. Whilc CD33
antibodics bind to virtually all GM colony-forming cells."
thcy do not bind to carlicr stcm cclls, whcrcas CD34
MoAbs bind to both." Once again, singlc- and doublcpositivc populations with distinctivc light scattcr charactcristics wcrc obscrvcd. Thc YBS.BX'KD33- cclls (Fig 2A)
wcrc prcdominantly locatcd in thc blast ccll rcgion similar
to thc YBS.B8'/CD34' cclls (SCC Fig IB), whilc thc doublcpositivc cclls wcrc mostly in thc high forward and sidc
scattcr population of putativc dcvcloping mast cclls (Fig
2B). Monocytes and a proportion of cclls in thc lymphcvtc
and blast ccll rcgions had thc phcnotypc YBS.B8-/CD33'
(Fig 2C).
As an indication of whcthcr c-kit is also cxprcsscd by
committcd lymphoid progcnitors, thc cocxprcssion of this
markcr with CDlO (common acutc lymphoblastic lcukcmia
antigcn, CALLA) was cxamincd using MoAb JS. In spccimcns from thrcc normal donors, no ovcrlap bctwccn thc
two markcrs was sccn aftcr correction for thc ncgativc
control (Sal-2 in placc of YBS.B8). Furthcrmorc. thc fcw
cclls in thc doublc-positive rcgion had apparently random
scattcr charactcristics (Fig 3B). In contrast, thc singlc-
positivc cclls had thc cxpectcd scattcr charactcristics (Fig 3,
A and C). In thc singlc cxpcrimcnt in which cocxprcssion of
CD34 with CD33 and CDlO was studicd, ovcrlapping but
nonidcntical populations of positive cclls were obscrvcd in
both cases as cxpcctcd. Rcsults for all two-color analyscs
arc summarizcd in Tablc I.
Expression of c-kit hv erythroid and multipotential myeloid
progenitorcell.$. Wc prcvious1yshowcd'"that MoAb YBS.BX
could bc uscd to isolatc a proportion of thc colony-forming
cclls from normal bonc marrow using an immunc rosctting
tcchniquc. Although only about 50% of the CFU wcrc
obtaincd in thc positivc fraction (compared with about 90%
with a CD34 MoAb using thc samc tcchniquc), thcsc
cncompasscd all typcs of committcd mycloid progcnitors.
Thcsc cxpcrimcnts havc now bccn cxtcndcd to cxaminc thc
cxprcssion of c-kit by crythroid (CFU-c and BFU-c) and
multipotcntial mycloid progcnitors (CFU-mix). Lowdcnsity nonadhcrcnt bonc marrow cclls from two normal
donors wcrc fractionated by immunc rosctting using MoAbs YBS.B8, BI-3CS. or Sal-2 and plated in methyl ccllulosc culturcs with PHA-LCM and crythropoictin.
The rcsults of onc cxpcrimcnt arc shown in Tablc 2;
A
a
l
:
m
>-
2
LL
.......
:
............
.
. . ..........
. ....
. . .' . e ...
. . . ..........
.. ... .
5:cn
,.,
*
. ...--..4. ,
............
..
i
. ..-..t+..
. . '...*Y. ;.::4:;:
.. 1 . - , ,!.,
*.- ,
I
"
kl
C
,
.
.
.I
.
.
FSC
C
I
Fig 2 Pmem of coexpresrion of E-kif and CD33
antigen on normal human bone marrow cells. Cells
were labeled with MoAbs YBS.68 (IgG1) and My.9
(IgGZb). Other details were as for Fig 1 except that
fluorescein-labeled anti-lgG2b instead of anti-lgG2a
was used. (A) YB5.B8 single-positive cells highlighted; (6) double-positive cells highlighted; (C)
CD33 slngle-positive cells highlighted.
,.
v)
v)
DL
.
. .
.- ..: ....
.*)
0
FSC
_.a.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
34
ASHMAN ET AL
A
a3
m
10
.:.. .
......
. .... .
;..."
..
M/
m
>-
9
L
. . r
v)
0'
FSC
B
C
Fig 3. Lack of coexpression of c-kit and CDlO
antigen on normal human bone marrow cells. Cells
were labeled with MoAbs YB5.88 (lgG1) and J5
(IgGzcl). Other details were as for Fig 1. (A) YB5.B8
single-positive cells; (B) apparent double-positive
cells; (C) 55 single-positive cells.
similar rcsults were obtained in the other. In these expcrimcnts, YB5.B8 was similar in cficiency to the CD34 MoAb
BI-3C5 in isolating CFUc, CFU-e, BFU-e, and CFU-mix.
Thus, it can be concludcd that thc majority of crythroid and
Table 1. Pattems of Coexpressionof c-kit Detected with MoAb
YB5.B8, and Other Progenitor Cell Markers, CD34, CD33,
and CDlO Antigens
% of Low-Density Bone Marrow Cells: Median h"
n=8
N=3
n=3
n=1
n=1
Y05.08'1CD34
2.9 (0.8-4.5)
Y05.08'1CD33
1.4 (1.0-3.9)
Y05.08'lCALLA'
2.2 (2.2-8.1)
CD34'1CD330.9
CD34'1CALLA1.o
Y05.08'ICD34'
1.6 (0.4-3.0)
Y05.08'1CD33'
1.6 (1.0-4.4)
Y05.08'1CALLA'
0
CD34'1CD33'
0.6
CD34'1CALLA'
0.4
Y05.08 /CD34'
0.5 (0.05-1.2)
Y05.08 lCD33'
23.2 (5.5-26.4)
Y05.08 ICALLA'
4.1 (1.1-8.4)
CD34-lCD33'
6.0
CD34 ICALLA1.o
Summary of two-color immunofluorescence data on normal iowdensity bone marrow cells. In each case n indicates the number of
independentspecimens.
multipotential myeloid progcnitors cxpress c-kif as well as
the CD34 antigcn.
DISCUSSION
Thc availability of an MoAb, YB5.B8, which is specific
for thc cxtraccllular domain of thc human c-kit growth
factor rcccptor, has cnablcd us to cxaminc thc ccllular
distribution of thc rcccptor in normal bonc marrow. Prcvious studies of c-kif cxprcssion in murinc" and human
normal and leukemicUmarrow have used probes for c-kit
mRNA in Northcrn analysis, and thus have been rcstrictcd
to thc cxamination of wholc populations rather than individual cclls. In this study of bonc marrow aspirates from 11
hematologically normal adult donors, wc have defined a
rcfcrcncc range for thc proportion of cells expressing c-kif
and defined their two-dimcnsional light scatter characteristics by flow cytomctry. Monocytes, maturing cells of thc
neutrophil lincagc, and thc bulk of cells in the lymphocyte
rcgion f d c d to bind thc YB5.B8 antibody. The positivc
cclls were in the blast ccll rcgion, or wcrc largc cclls, many
of which had high sidc scattcr and may be maturing mast
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C-KIT EXPRESSION IN NORMAL BONE MARROW
35
Table 2. Expression of c-kit and CD34 by Hematopoietic Progenitor Cells
Mean No. of ColoniesiPlate ? SEM (23replicates)
CD34
+
-
YB5.68
+
-
Sal-2
+
-
CFU-c
CFU-e
BFU-e
37.6 f 0.9
4.6 10.4
11.0 f 0.6
3.2 f 0.3
11.3 f 0.6
2.9 10.3
1.8 f 0.3
11.5 f 0.5
9.6 f 0.8
1.5 10.2
9.1 f 0.5
1.4 f 0.3
0.9 f 0.2
10,l -c 0.6
38.8 f 0.7
1.4 $ 0.2
4.7 f 0.4
37.9 f 0.7
CFU-mix
0.10
0.0
0.10
0.0
0.0
0.17
f 0.07
f 0.0
f 0.07
f 0.0
f 0.0
10.08
Total
58.3 f 1.5
9.4 f 0.7
59.3 f 1.1
5.6 f 0.4
7.3 f 0.6
59.7 1 1 . 1
Low-density nonadherent cells from a bone marrow aspirate from a healthy normal volunteer were fractionated using the indicated MoAbs by
immune rosetting as described in Materials and Methods. After fractionation, the cells were counted and, for each antibody, the positive and negative
frac;ions were resuspended in equal volumes such that each plate received the positive or negative fraction from 5 x lo4cells. MoAbs used were
BI-3C5 ((334); YB5.68 (anti-c-ht), and Sal-2 (negativecontrol). All antibodies are of lgGl isotype.
cells. The mean percent of low-density bone marrow cells
that bound the YB5.B8 antibody was 4.0 ? 1.8. The upper
limit of normal (mean + 2 SD) is 7.6% based on this
sample. We have previously reported that the expression of
relatively bigh levels of the antigen detected by MoAb
YB5.B8 on peripheral blood blast cells is strongly predictive
of a poor response to standard chemotherapy in AML.3'
Characterization of the expression of c-kit by normal bone
marrow populations is of central importance to the refinement of diagnostic tests based on the use of this marker.
The kinds of cells in normal bone marrow that express
c-kit and respond to the KL might be predicted from the
nature of the defects observed in W and S1 mutant mice.
While these mice display defective hematopoietic stem cell
and stromal cell function, respectively, in
the erythroid and mast cell lineages are most profoundly affe~ted'~.''~~'
in vivo. Therefore, it was not surprising to find
that the majority of BFU-e and CFU-e as well as CFU-mix
could be isolated in the YB5.BS-positive fraction of normal
human bone marrow. We have previously reported that at
least a proportion of bi- and uni-potential nonerythroid
progenitors could also be isolated with this antibody.16
Reliable culture techniques for human mast cell differentiation in vitro have recently been d e s ~ r i b e d . ~This
~ . ~ "antibody provides a means of determining at what stages of
mast cell development c-kit expression occurs. It is also of
obvious interest to determine whether long-term bone
marrow culture-initiating cells express c-kit, especially in
view of the dependence of these cultures on interactions
between stromal and stem cell^.^^,^^
The pattern of coexpression of c-kit with the prototype
hematopoietic stem/progenitor cell marker, CD34, was
investigated. The CD34 antigen is present on all committed
myeloid progenitor cells capable of producing colonies of
differentiated cells in vitro (CFU)3043,44
as well as early
progenitors that subsequently give rise to CFU in cult~re:~,~'Purified CD34-positive cells have been used to
bring about hematopoietic reconstitution of lethally irradiated baboons,* indicating that pluripotential stem cells
express this marker. The lack of reliable culture systems for
the study of human (as distinct from murine) lymphopoiesis
has hampered assessment of the expression of CD34 by
lymphoid progenitors. However, consistent with its presence on both myeloid and lymphoid normal progenitor
cells, the CD34 antigen bas been demonstrated on both
lymphoblastic and myeloid leukemic cells, as well as in
lymphoid blast crises of chronic granulocytic le~kemia.'~
Furthermore, putative B-lymphocyte precursors have been
demonstrated to be CD34-positive:'
MoAb YB5.B8 and the CD34 antibody ICH-3 bound to
overlapping but nonidentical populations in normal bone
marrow. The proportion of CD34-positive cells that also
bound YB5.B8 was 77% ? 12% (eight experiments). These
cells were located in a region of the two-dimensional
scatter-gram that appeared to overlap those identified by
Andrews et a148,49
as containing the CD34-positive cells that
directly or indirectly give rise to colonies in vitro, ie,
respectively, the committed myeloid progenitor pool, and
its precursors. Consistent with the interpretation that c-kit
is expressed on the precursors of CFU as well as CFU
themselves, many of the YB5.B8' cells in the blast cell
region of the two-dimensional light-scatter plot, especially
those with lower forward scatter, did not bind the CD33
MoAb (Fig 2A). The CD33 antigen is present on the
majority of CFU but absent from their p r e c ~ r s o r s . ~ ~ ~ ~ '
While culture of sorted cells under long-term stromadependent bone marrow culture conditions is necessary to
confirm that early progenitor cells are YB5.B8+,the biology
of the W/Sl system in the mouse (see above) predicts that
these cells would express c-kit. Furthermore, the KL, also
known as stem cell factor, was shown to synergize with
other factors in the growth of murine high-proliferative
potential colony-forming cells, which are believed to be
hematopoietic stem cells?" However, it must be noted that
YB5.B8+/CD34+ cells make up 1.5% & 1.0% of all lowdensity bone marrow cells, and are therefore too numerous
to be a pure population of hematopoietic stem and progenitor cells. As was shown in the murine system?l selection
based on multiple markers including lineage-specific antigens to eliminate more mature C ~ I I S and
, ~ ~ other markers
such as CD45 is~forms,'~
is necessary to obtain pure stem
and progenitor cell populations.
Many of the CD34-positive, YB5.Bg-negative cells were
located in the lymphocyte region of the scatter plot, and
thus may correspond to the committed early lymphoid cells
that coexpress the CDlO a n t i g e ~ ~This
. ~ ~was
' ~ ~consistent
with the distribution of the CDlO+/YBS.B8- cells seen in
two-color immunofluorescence experiments. The failure to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
36
ASHMAN ET AL
observe cells binding both the CDlO antibody and YBS.B8
suggests that human lymphoid precursor cells do not
express c-kit. Consistent with this, we did not observe
YB5.BS binding to any lymphoid leukemia^,'^ and Andre et
al” did not detect c-kit mRNA in a series of lymphoblastic
cell lines. However, stem cell factor synergizes with IL-7 in
stimulating murine pre-B cell colony f~rmation,’~
indicating
a role for c-kit in murine lymphopoiesis. Whether this
indicates a species difference, or an extremely restricted
stage of expression of c-kit in both murine and human
lymphopoiesis remains to be determined, as our data do not
rule out the coexpression of c-kit and CDlO on a very small
proportion of the normal bone marrow cells ( < -0.2%).
In summary, c-kit is expressed by approximately 4% of
low-density cells in normal bone marrow. These can be
divided into three categories based on surface phenotype
and light-scatter characteristics. One population of medium
sized cells coexpressed the CD34, but not the CD33
antigen, and may include early hematopoietic progenitor
cells, although this requires confirmation by direct culture.
Another population of larger cells with low side scatter
coexpressed both the CD33 and CD34 antigens. These cells
probably include multipotential and lineage-restricted progenitors capable of direct colony formation in semisolid
media. Direct evidence that these CFU express c-kit has
been provided. The third group of kit-positive cells were
large cells with high light scatter, coexpressed CD33 but not
CD34, and may be developing mast cells. No evidence was
found for the expression of c-kit by CD10+ early lymphoid
cells. MoAbs, such as YB5.B8, which identify c-kit will be a
valuable addition to MoAbs to other surface markers such
as the CD33 and CD34 antigens in defining the hierarchy of
human hematopoietic progenitor cells.
ACKNOWLEDGMENT
We thank Pam Dyson for assistance with the CFU-mix assay, and
Dr Robert Tindle for the gift of MoAb BI-3CS.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1991 78: 30-37
Expression of the YB5.B8 antigen (c-kit proto-oncogene product) in
normal human bone marrow
LK Ashman, AC Cambareri, LB To, RJ Levinsky and CA Juttner
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