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Granulomonocyte-Associated Lysosomal Protein Expression During In Vitro
Expansion and Differentiation of CD34+ Hematopoietic Progenitor Cells
By Clemens Scheinecker, Herbert Strobl, Gerhard Fritsch, Bettina Csmarits, Otto Krieger, Otto Majdic,
and Walter Knapp
Using an in vitro expansion and differentiation system for
human CD34+ cord blood(CB) progenitor cells, we analyzed
the induction and expression kinetics of the granulomonocyte associated lysosomal proteins myeloperoxidase (MPO),
lysozyme (LZ), lactoferrin (LF), and macrosialin (CD68).
Freshly isolated CD34+ CBcells were negative for LZ and LF,
and only small proportions expressed MP0 (496 f 2%) or
CD68 (3% f 1%). Culturing of CD34+ cells for 14 days with
interleukin (lL)-l, IL-3,IL-6, stem cell factor, granulocytemacrophage colony-stimulating factor (GM-CSF), and G-CSF
resulted in onaverage a 1,750-fold amplification of cellnumber, of which 83% f 7% were MPO+. Without addition of
GM-CSF and G-CSF, lower increases in total cell numbers
(mean, 21l-fold) and lower proportions of MPO’ cells (54%
f 11%) were observed. The proportion of MPO+cells slightly
exceeded but clearly correlated with the proportionof cells
positive for thegranulomonocyte associated surface mole-
cules C D l l b (Mac-l), CD15 (Lex), CD64 (FcyRI) CD66, or
CD89(FccuR). At day 14 MPO+ and LZ+ cells were virtually
identical. However, at earlier time points during culture
(days 4 and 71, single MPO+ or LZ+ cell populations were
also observed, which only later acquired LZ and MPO, respectively. Maturation ofcells into theneutrophilic pathway
was indicated by theacquisition of MPO, followed byLZ. In
contrast, maturation of cells into the monocytic pathway
was indicated by the acquisition of LZ followed by MP0
and CD14. CD68 was found t o be expressed at day 4 by the
majority of cells and was notrestricted t o t h egranulomonocytic cells, as cells with megakariocytic (CD4l+)or erythroid
(CD71hi) features were CD68+. LF expression was observed
only in GM- plus G-CSF-supplemented cultures, in which
only 26% f 5% of cells expressed LF by day 14.
0 7995 by The American Society of Hematology.
G
These observations prompted us to analyze in more detail
the kinetics of MP0 expression during granulomonocyte development and to include additional granulomonocyte-associated lysosomal proteins such as LZ, LF, and CD68 in our
studies. Cord blood (CB) CD34’ cells are an ideal model
for these studies. Such isolated cells tend to be more immature than CD34’ cells from
and were found by
us
to contain only small numbers of MPO’ cells.I4Additionally,
they can be easily cultured in vitro and, with appropriate
cytokines, be induced to proliferate and differentiate in a
coordinate manner. Thus, we were able to follow, in vitro,
the individual stages of granulomonocyte differentiation
from immature CD34’MPO- cells to MPO’LF- and
MPO+LF+ or MPOfCD14’ cells, respectively and to relate
the expression of M P 0 and LF to the expression of LZ and
CD68 and a variety of informative surface marker molecules.
RANULOMONOCYTIC cells play a key role in host
defense and have a critical involvement in inflammatory processes. Unlike B and T lymphocytes, mature granulomonocytic cells lack clonal antigen-specific receptor molecules. However, they possess a variety of monomorphic
recognition structures in which they can recognize invading
microorganisms or altered self structures. Moreover, mature
granulomonocytic cells are equipped with a number of specialized bioactive agents, such as digestive enzymes and
cytotoxic polypeptides, which are essential to the defense
against infection. Among these agents are myeloperoxidase
(MPO), lysozyme (LZ), lactoferrin (LF), and macrosialin
(CD68). MPO, which is a major constituent of primary neutrophil granules and monocyte lysosomes’**plays an important role in oxygen-dependent microbicidial effector
function^.^ LF, an iron binding bactericidalhacteriostatic
protein4is selectively localized in secondary granules of neutrophil granulocyte^.^ LZ, a bactericidal molecule with the
capacity to cleave bacterial peptidoglycans6is present in both
granulocytes and monocytes and localized in primary, as
well as secondary, granules of neutrophil granulocyte^.^.^
CD68, a recently characterized lysosmal/plasma membrane
shuttling protein with unknown function,839
is widely used by
immunohistologists as a pan-macrophage marker molecule.
Both the sequential appearance of lysosmal proteins during
granulomonocytic differentiati~n’~~.~.’O
and the expression patterns of granulomonocyte-associated surface molecules during
granulomonocyte development from immature hematopoietic
precursor
are well
described. So far, little is
known
ofhow the kinetics of lysosomal protein expression during
early stages of differentiation are related to the expression of
established differentiation-linked surface antigens.
Using flow cytometry together with
a suspension staining
technique for the detection of intracellular MP0 and monoclonal antibodies (MoAbs) against MPO,I3 we have been able
to demonstrate that MP0 andorits proform is expressed very
early in myeloid differentiation and can be found in
a substantialproportion(35%
+. 9%) of CD34+ bonemarrow(BM)
cells, which coexpress CD45RA and which possess in vitro
clonogenic ~ 0 t e n t i d . l ~
Blood, Vol 86,No 1 1 (December l), 1995:pp 4115-4123
MATERIALS AND METHODS
Antibodies
Antibodieslfluorochrome conjugates, specific for the following
molecules were used in our studies: Myeloperoxidase (clone H-435), lactofenin (clone 4C5), lysozyme (clone LZ-l), CD117 (c-kit,
From the Institute of Immunology, Vienna International Research
Cooperation Center at SFI, University of Vienna, Vienna; CCRI, St
Anna Children’s Hospital, Vienna; Elisabethinen Hospital, DepartmentofInternal
Medicine I, Linz; and Institute of Immunology,
University of Vienna, Vienna, Austria.
Submitted February S, 1995; accepted August I , 1995.
Supported by Fonds zur Forderung derwissenschaflichen
Forschungin Osterreich and COMAC-BIO European Concerted
Action Program.
Address reprint requests to WalterKnapp, MD, Institute of Immunology, Borschkegasse 8A, A-1090 Vienna, Austria.
The publication costsof this article weredefrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I S U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/861I -0OOS$3.00/0
41 15
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41 16
clone 95C3), and CD14 (clone MEMI8) from An der Grub (A2572 Kaumberg, Austria); CD68 (clone Ki-M7) from Behring AG
(Marburg, Germany); CD34 (clone HPCA2), HLA-DR (clone L243),
CD38 (clone HB-7), CDla (clone Leu-6) from Becton Dickinson
(Mountain View, CA); CD33 (clone MY9) and CD13 (clone MY7)
from Coulter C o p (Hialeah, FL);CDw90 (Thy-l, clone 5E10) from
Pharmingen (San Diego, CA);CD64(FcyRI,
clone 32.2) from
ATCC (Rockville, MD); CD1 Ib(Mac-l, clone VIM12). CD15
(Lex, cloneVIMDS), CD16 (FcyRIII,clone VIFcRIII), CD71 (TW,
clone VIPI), CD41 (GPIIblIIIa, clone VIPLI), CD7 (clone CD76B7) from our laboratory; CD66a,c,d,e (clone CLB-gradlo) and
CD66b (clone B13.9/) from the IVth Workshop on Human Leukocyte Differentiation Antigens (Vienna, Austria); CD89 (FccuR, clone
A-62) kindly provided by M. Cooper (Birmingham, AL) and CD19
(clone HD37) kindly provided by B. Dorken (Heidelberg, Germany),
and CD45RA (clone MEM93) kindly provided by V. Horejsi (Praha,
Czech Republic).
IrnrnunoJluorescence Staining Procedures
Membrane staining. For membrane staining, cells (107/mL)
were incubated for 15 minutes at 0 to 4°C with conjugated (fluorescein [FITC] or phycoerythrin [PE]) or unconjugated MoAb. For
stainings using unconjugated MoAbs, FITC-conjugated F(ab'), fragments of sheep antimouse immunoglobulin antibodies (SAM) (An
der Grub) were used as a second step reagent as described previously.'* For triple stainings, cells were incubated with a biotin
conjugated primary antibody, and subsequently incubated with the
second step reagent streptavidin PerCP (Becton Dickinson Immunocytometry System, San Jose, CA). Cells were then subjected to intracellular staining (as described below) or analyzed directly byRow
cytometry (FACS).
Intracellular staining. For suspension stainings of intracellular
antigens, we used the reagent combination Fix&Perm from An der
Briefly, cells are first fixed in FixaGrub as described previo~sly.'~
tion Medium for 15 minutes at room temperature and after one
washing step, cells are resuspended and mixed with Permeabilization
Medium plus fluorochrome (FITC or PE) labeled antibody. After a
further incubation for 15 minutes atroom temperature, cells are
washed again and analyzed.
Flow cytometry. Flow cytometry analysis was performed using
a FACScan Flow cytometer (Becton Dickinson Immunocytometry
System).
CB Cells
CB samples were collected during normal full-term deliveries.
Mononuclear cells (MNC) were isolated within 6 hours after collection using discontinuous FicolVHypaque (Pharmacia, Uppsala, SWeden) density gradient centrifugation.
Depletion of Mature Cells
To enrich for immature progenitors, mature (CD34-l cells were
removed from CB MNC samples by one to two rounds of immunomagnetic depletion, as previously de~cribed.'~
For this purpose,
MNC were incubated for 40minutes at 4°Cwith a cocktail of MoAbs
specific to CD3, CD1 lb, CD14, CD16, CD20, and anti-GlycophorinA. Afterwards, cells were incubated with antimouse Ig coated magnetic beads (2 X lo7 beads per lo7 cells, Dynabeads; Dynal, Hamburg, Germany). Dynabeads, together with bound cells, were then
removed with a magnet. This enriched the population of CD34+
cells from 1.2% ? 0.8% to 15% ? 9%.
SCHEINECKER ET AL
CD34 MoAb HPCA2 (PE) and sorted on a FACSVantage (Becton
Dickinson Immunocytometry Systems, Mountain View. CA). Cells
were first selected for low vertical light scatter properties and then
sorted for expression of the CD34 antigen. The final ccll population
contained 90% to 98% (mean, 94%) CD34' cells.
Mugnetic cell sorting (MACS). PE-labeled CD34' cells obtained
after FACS sorting (as described earlier) precluded double and triple
stainings of isolated cells at early time points during culture. Therefore, CD34' cells were alternatively isolated from CB MNC using
the MACS CD34 Progenitor Cell Isolation Kit (Milteny Biotec,
Bergisch Gladbach, Germany), according to the instructions of the
manufacturer. The purity of the CD34" population ranged from 8 7 9
to 98% (mean, 94%). Virtually identical expansion rates and kinetics
of antigen expression were observed for CD34' cells obtained from
both separation procedures (data not shown).
Cultivation of CD34' CB Cells
Isolated CD34' CB cells were cultured in 24-well plates (Costar,
Cambridge, MA) (1 X lo4cells in 1 mL/well) in RPM1 1640 supplemented with L-glutamine (2.5 mrnoVL), penicillin (125 IWmL),
streptomycin (125 ug/mL), and pooled CB plasma (10%) at37°C
in a humidified atmosphere and in the presence of 5 8 CO2. For
growth induction, combinations of recombinant human cytokines
were used. Medium A contained interleukin-l (rhIL-l; 100 U/mL;
Sandoz, Basel, Switzerland), rhIL-3 (100 UlmL; Behring AG, Marburg, Germany), rhlL-6 (10 ng/mL; Sandoz), and stem cell factor
(rhSCF;20 ng/mL; Amgen, Thousand Oaks, CA). Medium A +
GM/G was Medium A supplemented with granulocyte-macrophage
colony-stimulating factor (rhGM-CSF; 100 ng/mL; Sandoz) and
granulocyte colony stimulating factor (rhG-CSF; 100 U/mL, Behring
AG).
Morphology of Cells
Freshly isolated CD34' CB cells and cells cultured for 4, 7, or
I4 days were cytocentrifuged on microscope slides using a Cytospin2 centrifuge (Shandon Southern Products, Ltd, Astmoor, UK),
stained with May-Griinwald-Giemsa, and analyzed by light microsCOPY.
Colony Assays
At days 0, 7, and 14 after initiation of the suspension culture,
aliquots of cells were analyzed for their colony forming capacity in
a methylcellulose-based semisolid culture mediumI9 containing 2.5
U/mL of recombinant human erythropoietin (rhEpo; Cilag, Schaffhausen, Switzerland), 100 U/mL of rhGM-CSF (Genzyme, Boston,
MA), I O U/mL rhIL-3 (Genzyme) and 20 ng/mL rhSCF (Amgen).
One milliliter of soft gel was used per 35-mm dish, and each test
was performed in duplicate. The cell density in the culture medium
ranged from 50 to 2.100/mL. At least three different cell concentrations were plated for each sample. The cultures were incubated at
37°C in a humidified atmosphere and in the presence of 5% COz
and 3% 0, in N,. The numbers of colony-forming units (CFU, >50
cells per colony) and cell clusters (10 to 50 cells per colony) were
evaluated on day 14.
The absolute number of cells with colony-forming capacity (CFC)
grown at each time point during the suspension culture was calculated by multiplying their incidence (per cells seeded in methylcellulose) by the number of nucleated cells present in each suspension
culture.
RESULTS
Isolation of CD34' CB Cells
Phenotypic Characteristics of Fresh CD34' CB Cells
Fluorescence-activated cell sorting. CB MNC enriched for immature progenitor cells by negative depletion were stained with anti-
The expression pattern of lysosomal proteins in CD34'
CB cells is shown in Table 1. At day 0, only small propor-
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A117
LYSOSOMALPROTEINEXPRESSION
tions of CD34+ CB cells expressed M P 0 (4% 2 2%) or
CD68 (3% 2 1%) and virtually all of them were negative
for LZ or LF expression. For M P 0 and CD68, this is in
sharp contrast to the situation in bone marrow, where M P 0
and/or CD68 expression can be found in about 20% to 30%
of CD34' ~ e 1 l s . lLF
~ expression cannot be detected in
CD34+ BM cells (unpublished observation), for LZ, no data
for BM CD34+ cells are available.
The surface molecule distribution patterns of CD34+ CB
cells (Table 2, day 0) were similar to those previously reported by other g r o u p ~ . " ~The
~ ~ ~majority
~'
(>80%) of
CD34' CB cells were found to express HLA-DR, CD38
and/or CD1 17 (c-kit). A subset expressed CD45RA (15% 2
8%), and only a small proportion were Thy-l positive (4%
? 4%).
CD33 and CD13 were found to be expressed in up to
52% and 60% of cells, respectively. Expression levels were
generally low and highly variable from sample to sample.
Expression of the granulomonocyte-associated surface
molecules C D l l b (Mac-l), CD14, CD15 (Lex), CD16
(FcyRIII), CD64 (FcyRI), CD66, and CD89 (FcaR) was
not detectable, or were cells with megakariocytic (CD41) or
lymphoid (CD7, CD19) features found. Transfemn receptor
molecules (CD71), thought to be associated with erythroid
differentiation22were found on a small subset of CD34+
cells.
Growth Pattern of CD34+ CB Cells in Suspension Culture
In vitro culture of purified CD34' CB cells in either Medium A (containing the cytokines IL- I , IL-3, L-6, and SCF)
or Medium A + GM/G (further supplemented with the cytokines GM-CSF and G-CSF) led to significant cell growth.
As can be seenin Fig l , the total cell numbers increased
with Medium A 20 5 14-fold in the first 7 days and 211 2
140-fold from day 0 to day 14. With Medium A + GM/G
the total cell number increases were even more pronounced.
In the first 7 days, numbers increased 32 2 24-fold and from
day 0 to 14, a 1,753
1,140-fold increase of total cell
numbers was observed.
Table 2. Kinetics of the Expression of Surface Receptor Molecules
During Culture of CD34' CB Cells
Medium A
CD34
CD1 17(c-kit)
CDw9O (Thy-l)
HLA-DR
CD38
CD45RA
CD33
CD13
CD14
CD41
CD71h'
CD7 & CD19
CDla
Medium A + GMIG
CD34
CD1 17(c-kit)
CDMO (Thy-l
HLA-DR
CD38
CD45RA
CD33
CD13
CD11b
CD14
CD15
CD16
CD64
CD66
CD89
CD41
CD71h'
CD7 & CD19
CDla
Dav 0
Dav 7
94 2 4
81 2 14
4 2 4
95 2 1
90 2 6
15 f 8
52 2 29
60 ? 20
<3
13
423
<3
<3
623
28 2 17
<3
75 2 6
90 2 4
7 2 4
70 2 29
61 ? 18
12 2 5
522
25 ? 7
<3
<3
94 2 4
81 2 14
4?4
95 ? 1
90 5 6
15 2 8
52 2 29
60 c 20
<3*
<3
13'
<3*
<3*
<3*
<3*
<3
423
<3
<3
3 2 1
18 t 2
<3
67 ? 9
92 2 5
7 2 5
77 2 25
76 f 18
NT
7 2 2
NT
NT
NT
NT
NT
423
17 2 8
<3
<3
Dav 14
<3
20 2 4
<3
35 2 7
82 2 6
7 2 4
58 2 32
54 2 14
19 2 4
523
18 2 3
<3
<3
C3
422
<3
14 If: 5
56 2 7
626
64 2 36
56 2 8
76 2 IO'
17 2 7
59 % 14*
4 2 3'
65 f 26'
66 f 16'
76 t 17"
<3
522
<3
13
Numbers indicate the mean percentage ? SD of positive cells in
four experiments unless otherwise indicated.
Abbreviation: NT, not tested.
* (n = 3).
CFC of In Vitro Expanded Cells
During the first 7 days of culture, the total number of cells
with CFC increased almost in parallel with the total numTable 1. Kinetics of the Expression of Lysosmal Proteins During
Culture of CD34' CB Cells
Day0
Day 4
Day 7
14 Day
Medium A
MP0
4 2 2'
Lz
<3'
LF
<3*
CD68
321'
Medium A + GMIG
MP0 8 3 2674 241482 1 24 2 2 '
Lz
<3'
LF
<3*
CD68
683
8962
021
t18'1
54 2 11
30 2
4910
2 12
34
55
4
23
2
12
1
71
22
<3
<3
75216
78210
523
7529
363722121217112
<3
26 2 5
<3
Numbers indicate the mean percentage -C SD of positive cells in
four experiments unless otherwise indicated,
* (n = 6).
bers of all cells in suspension culture. The proportion of
CFC decreased only slightly from 42% 5 16% at day 0
to 33% 2 8% (Medium A) and 31% ? 18% (Medium A
+ GM/G) atday 7. In the second week of culture, however,
the proportion of CFC fell in Medium A to 7% ? 3% and
in Medium A + G W G to 5% 2 2% of all cells in suspension culture.
Moreover, the types of colonies formed changed during
culture. The proportion of CFU-GM increased in Medium
A + GM/G at the expense of burst-forming unit-erythroid
(BFU-E) and CFU-MIX from 59% t 17% at day 0 to 92%
-C 7% at day 14. In Medium A, 78% ? 18% of day 14
colonies originated from CFU-GM. Paired analysis of individual CD34+ CB populations showed that absolute numbers
of CFU-GM colonies at day 14 were on average fivefold
higher (4 to 6) in Medium A + GM/G than compared with
Medium A. That differentiation of the majority of cells occurred was further substantiated by flow cytometric analysis
of lysosmal and surface molecule expression.
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4118
SCHEINECKER ET AL
Medium A
l
.
,
0
.
,
4
Medium A+GM/G
.
I
7
14
0
DAYS IN CULTURE
Phenotypic Changes During In Vitro Expansion
Several dramatic changes in lysosmal and surface molecule expression were observed during in vitro culture of
CD34+ CB cells.
Induction of lysosmalprotein expression. As can be seen
in Table 1, the most rapidly induced lysosmal protein is
CD68. By day 4 of culture, the majority of cells expressed
CD68. At this time point, only a small subset of cells expressed M P 0 or LZ. LF expression is not detectable at day
4. At day 7, substantial proportions of cells express M P 0
and/or LZ. CD68 expression is maintained and LF expression is not yet detectable. At day 14, the proportions of cells
expressing M P 0 or LZ further increase and reach values of
83% ? 7% and 71% ? 12%, respectively in cultures grown
in Medium A + GWG. At day 14, LF expression can be
detected for the first time. In Medium A + GWG, 26% t
5% of cells are LF positive, as compared with 5% 5 3% in
Medium A.
Correlation of MP0 expression withLZand CD14expression. The similar proportions of MPO+ and LZ+ cells during culture first suggested that the same population of cells
coexpress M P 0 and LZ in a coordinate manner. However,
further combined analysis of M P 0 and LZ expression at
a single cell level showed distinct populations of cellular
expression levels of M P 0 and LZ. Most notably, at day 4,
there were LZdimMPO+,and LZh-”PO’ fractions. In addition, a subset of LZdimMPO-cells could be distinguished.
On further culture, LZhiMPO+,and LZamMPO+cells began
to merge at day 7, and on day 14, homogeneous staining for
both M P 0 and LZ could be detected in the majority of
cells (Fig 2 ) . Only in Medium A could the small subset of
LZdimMPO-cells still be detected on day 14.
To further analyze the nature of the two major subsets,
LZhiMpOdim and LZamMPOhi,triple staining experiments
were performed. As illustrated in Fig 3, the majority of gated
LZhiMpOdim cells coexpress CD14 and, therefore, would
seem to represent monocytic cells, whereas LZdimMPOhi
cells
are CD14 negative.
Correlation of CD68 expression with MPO, CD41, and
CD71 expression. CD68 expression preceded M P 0 or LZ
expression and the number of CD68+ cells clearly exceeded
the number of MPO+ or LZ’ cells during the first 7 days of
culture.
4
7
.
,
14
Fig 1. Growth pattern of
CD34’CB
cells in suspension
culture.Increases in total cell
numbers (01, absolute numbers
of CD34+ cells (A) and absolute
are
numbers of CFC’ I-)
shownduringsuspensionculture in Medium A and Medium A
+ GMIG. Numbers indicate the
mean number xE-3 f SE in four
experiments
unless
otherwise
indicated *In = 3).
This raised the question as to whether the CD68+MPOcells (found during the first 7 days of culture) represent
immature noncommitted progenitor cells, myeloid committed progenitors not yet expressing M P 0 or, progenitor cells
committed to other hematopoietic lineages, which are at later
culture stages overgrown by myeloid cells. To address this
question, we performed triple stainings for intracellular
CD68 and M P 0 and surface CD4 1or CD7 1. As can be seen
in Fig 4, on day 7 of culture 62% ? 19% of CD68’ cells
coexpress MPO. In addition, small subsets of CD41+CD68+
and of CD71iCD68+ cells can be detected. These CD41+
or CD71+ cells do not coexpress M P 0 and are, therefore,
probably committed to the megakariocytic (CD41+) and erythroid (CD71h’)lineage, respectively.
Changes in suflacemoleculeexpression.
Concomitant
with the induction of lysosmal protein expression, several
changes in surface molecule expression occurred. Most dramatic was a rapid downregulation of the progenitor cell associated surface molecules CD34,CD117 (c-kit), CDw90
(Thy-l) and, less pronounced, HLA-DR. In parallel, several
granulomonocytic differentiation antigens including CD I 1b
(Mac-l), CD14, CD15 (Lex), CD64 (FcyRI), CD66, and
CD89 (FcaR) started to be expressed. No significant changes
were observed for the surface molecules CD33 and CD13.
CD16 (FcyRIII) could only be detected on a small subset
of cells on day 14 of culture in Medium A + GWG. The
numbers of CD41’ and CD71h’cells increased considerably,
but their relative proportions remained relatively low (<3%
to 7% for CD41’ and <3% to 22% for CD71”’). Cells with
lymphoid characteristics (CD7 and CD19) were never detectable.
Changes in Cell Type and Morphology
Results of morphologic analyses of freshly isolated
CD34+ CB cells and of cells cultured for 4, 7, and 14 days
in Medium A and Medium A + GWG, respectively are
summarized in Fig 5.
Similar to both culture conditions, the mean percentage
of blast cells decreased from 100% at the start of culture to
95% t 1% (Medium A) and 91% 5 3% (Medium A + GM/
G ) at day 4 and further to 57% 5 6% (Medium A) and 57%
-C 2% (Medium A + GM/G) at day 7. At day 14, however,
18% t 3% of cells had blast cell morphology in Medium
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LYSOSOMAL PROTEIN EXPRESSION
4119
A compared with only 5% i 2% in Medium A + GM/
G. Promyelocytes were identified from day 4 onwards and
proportions peaked at day 7 (23% i 4% in Medium A and
25% -t 2% in Medium A + GM&). Theappearance of
myelocytes and metamyelocytes lagged behind promyelocytes and increased during culture to become the predominant (50% -t 10% in Medium A and 60% -t 8% in Medium
A + GM/G) cell type at day 14.
Monocytesormacrophageswerenotdetectablebefore
day 7 and represented 9% -t 2% of cells in Medium A and
18% 2 8% in Medium A + GM/G at day14.Veryfew
mature neutrophils (1% to 2%) were observed after 14 days.
Freshly isolated CD34' cells contained no visible granules. At day 4, most cells still had blast morphology, but a
subset of 6% t 2% contained some granules. M P 0 expres-
Medium 9
Medium A+GM/G
A
dO
I
I
?
d7
LZ
- NEG. CONTROL- -CD14
-
-CD14
-
Fig 3. Correlation of MP0 expression with L2 and CD14 expression. The dot plots showrepresentative triple stainings for MPO, LZ,
and CD14 at day 7 of suspension culture of CB CD34+cells in Medium
A + GMIG. The upper dot plotdisplays MP0 against L2 fluorescence
intensity. Quadrants were set according to isotype-matchednegative
control stainings. Arrows indicate increasing fluorescence intensity
displayed in logscale. The t w o distinct populations in terms of MP0
and L2 expression intensity (LZd""MPOhi= R1; and LZhiMPOdim= R2)
were gated and analyzed for theexpression of CD14. The histograms
showthat LZdimMPOhi cells areCD14-, whereas themajority of
LZhiMPOdimcells are CD14'as compared with the isotype-matched
negative control staining.
sion was found at day 0 and exceeded by far proportions of
granulatedcells (blastswithsmallscattered
granulesplus
promyelocytes) at day 4 of culture in Medium A + GM/G
(44% -t 12% v 14% % 3%, P = .01). This difference was
even more striking for the lysosomal protein CD68 (69% i
1I % positive cells on day 4 in Medium A GM/G). These
kinetics suggest that M P 0 expression precedes light microscopically detectable granulation of cells.
+
DISCUSSION
dl4
Fig 2. Induction of MP0 and L2 expression during suspension
culture of CD34' CB cells. The dot plots showrepresentative double
stainings for MP0 and L2 at days 0, 4, 7, and 14 during suspension
culture in Medium A and Medium+ GM/G,
A
respectively. Quadrants
were set according to isotype-matched negative control stainings.
Arrows indicate increasing fluorescence intensity displayed in log
scale.
In this study, the expressionkinetics during hematopoietic
differentiation of four granulomonocyte-associated lysosmal
proteins (MPO,LZ, CD68, and LF)were analyzed semiquantitatively at a single cell level using flow cytometry. As
a model system, we used in vitro culture and expansion and
differentiation of immature CD34+ hematopoietic progenitor
cells isolated from human CB.
Freshly isolated CB CD34+ cells have blast-likemorphology, the majority of them coexpresses c-kit and HLA-DR,
about 37% to 93% express CD13 and/or CD33, but they
lack expression of several granulomonocyte restricted surface molecules (Table 2), and the vast majority of them are
also negative for MPO, CD68, LZ, or LFprotein expression
(Table 1). On in vitro culture in the presence of appropriate
cytokines,wecouldexpandthesecellsup
to 3,400-fold
within 14 days and at the same time induce downregulation
of progenitor cell associated surface structures and upregulation of lineage associated structures. This allowed us tostudy
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SCHEINECKER ET AL
4120
-CONTROL
NEG.
CD68
--t
f
J
i
;
i
;
0
0
n
-
J
n
+
l
.(
MP0
__c
Fig 4. Correlation of CD68 expression with MPO,
CD41, and CD71 expression. The contour plots show
representative triple stainings for intracellular CD68
and M P 0 and surface CD41 or CD71, respectively,
at day 7 of suspension culture of CB CD34' cells in
Medium A GM/G. Onaverage 62% 2 19% of CD68+
cells coexpress MPO. In addition, small subsets of
CD41'CD68+ and CD71'CD68' cells can be detected.
CD4l+ or CD71+ cells are M P 0 . Quadrants were set
according to isotype-matchednegativecontrol stainings. Arrows indicate increasing fluorescence intensity displayedin log scale.
CD68
in parallel the induction kinetics and cellular distribution of
MPO, CD68, LZ, and LF protein expression.
We selected M P 0 as one of the lysosomalproteins to
study because it is certainly one of the best studied granulomonocyte restricted marker molecules.' Immunologic demonstration of M P 0 and/or its proform in cells has only recently becomepossibleand
i s now consideredthe most
specific and sensitive method for the identification of undifferentiated acute myeloid leukemia (AML) blast cells.11.21.24
Wehave been abletodemonstrate
that immunologically
detectable MPO, and/or its proform, are already expressed
at the CD34+ stageof normal hematopoietic progenitor cells
in human BM, but not, or only in very small numbers of
CD34' cells from CB or peripheral blood of adult^.'^
As a growth stimulus we used a combination of six cytokines (IL-I, IL-3, IL-6, SCF, GM-CSF, G-CSF = Medium
A + GMIG), previouslyused by Haylocket al" for the
generation of virtually pure granulomonocyte progeny and
compared this with asimilar stimulus not including GMCSF and G-CSF (Medium A) to preserve a more immature
phenotype.
We have shown that M P 0 expression is rapidly induced
on in vitro expansion and differentiation of CB CD34' cells
1) and, once inwith both cytokinecombinations(Table
duced, M P 0 is continuously upregulated. Comparative analysis of cell morphology showed that immunologically detectable M P 0 expression clearly
precedes
visible
granule
formation. At days 7 and 14, most cells cultured with Medium A + GM/G were strongly MPO+ and the proportion
of MPO+ cells clearly correlated with, although slightly exceeded, proportions of cells positive for the granulomonocyte associated surface molecules CD1 1b (Mac-l ), CD15
(Lex), CD64 (FcyRI), CD66, or CD89 (FccrR) (Table 2).
No suchcorrelationcould
be detectedbetween M P 0 and
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4121
LYSOSOMAL PROTEIN EXPRESSION
Medium A
Medium A+GM/G
I
0
4
7
14
0
4
7
14
DAYS IN CULTURE
Fig 5. Cell type and morphologyof cultured cells. Proportions of blasts, promyelocytes (promye), myelocyteslmetamyelocytes(myelmeta).
and monocyteslmacrophages (monolmacro) as assessed from May-Grunwald-Giemsa stained cytospin preparations. Numbers indicate the
mean percentage 5 SD of each cell type observed in three independent experiments from three individuals.
CD33 or CD13 expression. CD33 and CD1 3 were already
present on a substantial proportion of freshly isolated CD34'
cells and found to be
variably expressed, but not significantly
up or downregulated during culture.
Lysozyme representsanother lysosomal protein,which
within the hematopoietic system is selectively expressed by
cells of granulomonocytic
the
Comparative
analysis of LZ and M P 0 showed, particularly in the early stages
of granulomonocytic differentiation, two distinct
populations
in terms of M P 0 and LZ expression. This dichotomy may
represent an early dissociation between neutrophil development and monopoiesis (Figs 2 and 3). Cells committed to
differentiate along the monocytic lineage seem to arise from
a LZ+MPO- population and subsequently, after having entered a LZh'MPOd"" cell stage start to express CD14 molecules. Thus, LZ might precede M P 0 and CD14 expression
during monocytic differentiation. Neutrophil differentiation,
on the other hand, seems to
start with a MPO'LZ phenotype, then further develops to a MPOhiLZd"" expression pattern, and finally merges with the monocytic differentiation
pathway in a relatively homogenous MPOh'LZh'population.
This dissociation between M P 0 and LZ expression seems
to precede morphologicdifferentiation into either promyelocytes or promonocytes.It is detectableat day 4 of culture and
at this time point still 95% (Medium A) and91 % (Medium A
+ GM/G) of cells exhibit blast cell morphology. Very few
promyleocytes and virtually no monocytoid cells were detectable at this stage (Fig S).
A similar dichotomy in terms of M P 0 and LZ expression
in immature myeloid blast cells has also been observed in
leukemic cell samples. AML blasts with monocytic features
(FAB MS) were reported to be strongly reactive with antiL Z a n t i b o d i e ~ ~and
' . ~ ~weakly express MPO." On the other
hand, AMLblast cells lacking monocytic features (FABM I ,
M2, M3,) are not infrequently LZ""' or n e g a t i ~ e . * ~ - ~ '
Our findings with in vitro differentiated normal hematopoieticprogenitor cellsnowsuggest, thattheseparticular
expression patterns in AML samples mirror the situation in
normal myelopoietic differentiation. Monocytic precursors
seem, first, to synthesize LZ and only later MPO, while in
neutrophil development, M P 0 synthesis seemstoprecede
LZ. Differences between monocytes andneutrophils in their
regulation of LZ synthesis are also maintained at later stages
of development. Mature monocytes are thought to continuously synthesize and secrete LZ, while granulocytes do not
and only release the preformed e n ~ y m e . ~ ~ , ' ~
The CD68 molecule was included in our studies because
it is widelyusedin immunohistology asapan-monocyte/
macrophage markermolecule.33 Ofall the lysosmalproteins,
it was the most rapidly upregulated molecule, found in the
vast majority of cells at day 4 of culture. Quite clearly, not
all of these cells can be assigned to the monocyte/macrophage or the granulomonocytic lineagein general. They obviously also include other than granulomonocyte committed
cells, as we consistently observed CD68+ cells with megakaryocytic (CD41+) or erythroid (CD71h') features (Fig 4).
Consistentwith thisconclusion, CD68 expression has recently been reported in megakaryocytes""' and in erythroid
cell lines.' Selectivity was only detectable at a quantitative
level in that differentiated CD14+ cells at day were
14 clearly
characterized by the strongest CD68 expression of all cells
present in culture (data not shown).
LF was included as a marker molecule for the identification of a late stage in neutrophil development. LFis known
to be expressed first at the transition from the mitotic to
the maturation compartment in neutrophil de~eloprnent.~
In
agreement with that, LF is never foundin CD34' CB or BM
cells, or is LF detectable inblast cells from patientswith
acute myeloid leukemia^."^'^ Given the high cell amplification values (1,753 t 1,140-fold) in our cultures that account
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4122
SCHEINECKER ET AL
for at least 10 to 1 1 cell divisions, we were surprised to find
LF expression only at day 14 of culture and only in a minor
subset (26% 2 5%) of cells stimulated with Medium A +
GM/G. Even at day 14 of culture in Medium A + GWG,
the majority of cells (58% 5 4%) maintained a relatively
immature phenotype (MPOfLF-CD14-) that in normal BM
accounts for only a minor population of cells. These findings
are certainly not due to a methodologic problem in detecting
lactoferrin in our cell samples. With the flow cytometric
method used, lactoferrin is clearly detected in all neutrophils
from peripheral blood samples.39The in vitro differentiation
system used represents an ideal model to generate large numbers of immature granulomonocyte committed cells and to
analyze their molecular and functional characteristics.
ACKNOWLEDGMENT
We thank C. Bello-Fernandez, H. Stockinger, and S.A. Ali for
critically reading the manuscript and P. Buchinger for her help with
cell culture.
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1995 86: 4115-4123
Granulomonocyte-associated lysosomal protein expression during in
vitro expansion and differentiation of CD34+ hematopoietic
progenitor cells
C Scheinecker, H Strobl, G Fritsch, B Csmarits, O Krieger, O Majdic and W Knapp
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