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RAPID COMMUNICATION
Flt3 Ligand Induces Proliferation of Quiescent Human Bone Marrow
CD34+CD38- Cells and Maintains Progenitor Cells In Vitro
By Ami J. Shah, Elzbieta M. Smogorzewska, Charles Hannum, and Gay M. Crooks
Flt3 is a class 111 tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells
(HPC). In previous studies using stroma-free short term
assays, Ftt3 ligand (FL) has been shown t o induce proliferation of HPC at proportions similar t o or less than c-kit ligand
(steel factor, SF). Using long term stromal cocultivation
assays, w e studied the effects of FL on proliferation and
differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+CD38- cells. Cell
proliferation was significantly greater with FL than with SF,
when used individually or in combinations with interleukin3 (IL-3) andlor IL-6. The effect of FL was greater on bone
marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect
was seen with FL on more mature CD34+CD38+cells from
either BM or cord blood. The frequency of colony-forming
units (CFU) and high proliferative potential-colony forming
cells (HPP-CFC) during early culture (130 days) was increased by both SF and FL t o similar levels. However, in the
LTC-IC period (35 t o 60 days) and extended long-term culture
initiating cell (ELTC-IC) period (>60 days), the frequency of
CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL ( P < .0025). Fluorescenceactivated cell sorter analysis of cultures after 21 days
showed a significantly higher percentage of cells remained
CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S)
than in 3161s (0.7846 ? 0.5290 ~0.21%? 0.2990 respectively,
mean 2 SD). Cloning efficiency of BM CD34+CD38- cells was
significantly increased by the addition of FL t o the combination of 31615 (mean 11.7% vO.5%, P e .0001). These data
show that FL is able t o induce proliferation of CD34+CD38cells that are nonresponsive t o other early acting cytokines
and t o improve the maintainence of progenitors in vitro.
0 1996 b y The American Society of Hematology.
F
progenitors by their sustained clonogenicity in stromal cocultivation cultures." This sustained clonogenicity demonstrates enrichment of progenitor output from long-term culture-initiating cells (LTC-IC) and extended long-term
culture-initiating cells (ELTC-IC). In this report, we have
found that FL is highly synergistic with IL-3, IL-6, and
SF in inducing proliferation of CD34'CD38- BM cells and
results in expansion of colony-forming units (CFU) from
LTC-IC and ELTC-IC. In these long-term assays, the effects
of FL are significantly greater than with SF and are achieved
at least in part by increasing the proportion of quiescent cells
capable of responding to cytokine stimulation. In addition,
FL's effects on progenitor output in extended long-term culture may be also caused by an enhancement in the maintenance of progenitors in vitro.
LT3 IS A RECENTLY discovered member of the class
111 tyrosine kinase receptor family, which includes ckit and c - ~ s . ' In
- ~murine bone marrow (BM), Flt3 is preferentially expressed on primitive hematopoietic progenitor
cells (HPC) and is largely restricted in human hematopoietic
cells to the CD34+ progenitor populati~n.',~,~
The expression
of Flt3 on human and murine HPC has led to the hypothesis
that its ligand may have an important role in proliferation
of primitive hematopoietic progenitors.
Several murine studies have shown that Flt3 ligand (FL)
induces proliferation of highly purified HPC in synergism
with a number of other growth factors including interleukin6 (IL-6), IL-11, IL-12, granulocyte colony-stimulating factor
(G-CSF), and IL-3.5-7Studies of the effect of FL on human
cells have been more limited. FL had effects on human
CD34+ cells similar to those described in murine studies, ie,
alone or ill combination with IL-3, granulocyte-macrophage
CSF (GM-CSF), Pixy 321, or erythropoietin, FL caused cell
proliferation and enhanced colony
Muench et
a1," studying C D 3 4 T D 3 8 - h - human fetal liver cells,
found that FL acted in synergism with GM-CSF to induce
expansion of high proliferative potential colony-forming
cells (HPP-CFC), a highly primitive progenitor population.
Each of these reports used short-term (<21 days) liquid
suspension or semisolid cultures to examine the effect of
FL to induce proliferation of murine or human HPC. In all
comparisons with FL with another early acting cytokine steel
factor (SF), FL was either similar or inferior to SF either as
a single agent or in combination with other cytokines.
We report that a far more impressive proliferative response specific to FL is obtained when a primitive human
HPC population, CD34+CD38- cells, is studied during longterm culture on stromal support. Previous work has shown
that CD34TD38- BM cells are a primitive and highly quiescent population of cells with low cloning efficiency, even
when stimulated with a combination of the early acting cytokines IL-3, IL-6, and SF."." CD34TD38- cells are distinguished from the more mature and numerous CD34TD38'
Blood, Vol87, No 9 (May I),
1996: pp 3563-3570
MATERIALS AND METHODS
Cell sources. BM was obtained from consenting healthy adult
volunteers by aspiration from the posterior iliac crest. Umbilical
From the Divisions of Hematology/Oncology and of Research
Immunology and Bone Marrow Transplantation. Childrens Hospital
Los Angeles, Los Angeles; and DNAX, Palo Alto, CA.
Submitted December I , 1995; accepted January 31, 1996.
G.M.C. is the recipient of a Basil O'Connor Starter Scholar Research Award from the March of Dimes Birth Defects Foundation.
Supported in part by Grant No. ROI-HL-94-IO-B from the NHLBI
(G.M.C.).
Address reprint requests to Gay M. Crooks, MD, Division of
Research Immunology/Bone Marrow Transplantation, Childrens
Hospital of LAX Angeles, MS #62, 4650 Sunset Blvd, Los Angeles,
CA 9002 7.
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 1734 solely to
indicate this fact.
0 1996 by The American Soci&y of Hematology.
0006-497 I/96/8709-OO57$3.00/0
3563
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SHAH ET AL
3564
A
B
0
0
200
400
600
FSC-Height
800
1000
4
CDWFITC
Fig 1. FACS analysis of BM mononuclear cells (mnc) after preenrichment using the Miltenyi MACS device. (A) Forward scatter (size) and
side scatter (density) of B M mnc is shown. Region R1 is the "lymphoid gate" in which CD34* progenitor
cells are located. (B) CD34 and CD38
expression of BM cells from region R1. Quadrants are defined by FITC- and PE-labelled isotype controls (see Materials and Methods). Region
R2. used to define CD34TD38 cells for isolation, is defined as CD34' cells with PE-CD38 fluorescence less than half of the isotype control.
Region R3 was used to define CD34+CD38* cells.
cord blood wasohtainedafter
vaginal andcesareandeliveries
at
allysimilar with 72%homology at theaminoacid
level andare
Kaiser Permanente Hospital Sunset (Los Angeles. CA) after clamp- crossreactive
in their effects on mouse and human HPC."13 Recomhinant soluble mouse FL was produced in Escherichia coli. refolded.
ing and cutting of the cord. by drainage of blood into sterile collection tubescontaining the anticoagulantcitrate-phosphate-dextroseand
purified by sequentialanionandcationexchangechromatoused in all culturesata final concentration of 50
(Sigma, St Louis.MO). All BM andcord blood specimensweregraphies.FLwas
a unit of activity is defined as the amount needed for
obtained according to guidelines approved by the Childrens Hospital UhnL, where
Los Angeles on Committee Clinical Investigation. Cells were
prohalf maximal stimulation of 1 X IO" Baflt cells (a stable transformant
cessed within 24 hours of collection.
ofBa/F3cellsexpressing
the Flt3receptor). as described prelsolrrriorl of CD34" .srtl,poprrlrrtiorls by,~rrorc~.sc.erlc~c-r~cti~~r~tcrl
cell
viously." The concentration of 50 UlmL represents twice the minisortin,? (FACSI. Mononuclearcellswereisolatedfrom
BM and
mum concentration previously reported to result in maximal colony
cord blood using Ficoll Hypaque (Pharmacia. Piscataway. NJ) denformation of CD34-CD38-Lin- cells light-densityfetal liver cells."
sity centrifugation. The cells were subsequentlywashed with Hanks'
LnrlR-terrn srrorncrl crtltrtres and rnethylcellrrlose cultrrres. BM
Balanced Salt Solution (HBSS: GIBCO. Gaithersburg. MD). To enstroma was produced by culturing fresh BM mononuclear cells for
rich for CD34+ cells. low-density mononuclear cells were applied
at least 2weeks in stromalmedium(12.5%horseserum.
12.5%
to a mini-Magnetic Activated Cell Sorter column (mini-MACS:Milfetal calf serum [FCS: Gemini Bioproducts, Calabasas, CA], Iscove's
tenyi Biotec, Sunnyvale, CA). Using this method. purity of CD34Modified Dulbecco'sMedium[IMDM:GIBCOBRL,
Bethesda.
cell preparations ranged from 85% to 90%. Further purification of
1O-6 m o l L hydrocortisone
MD].
2-mercaptoethanol
[Sigma],
CD34'CD38-cellswasaccomplished
by staining the CD34* en[Sigma]. penicillidstreptomycin and glutamine). Macrophages were
riched populationwith fluorescein isothiocyanate(F1TC)-labeled
depleted from the stromal cultures by trypsinizing and replating at
anti-CD34 (HPCA2-FITC; Becton Dickinson [BDIS], San Jose. CA) least three times before final use in the long-term cultures. One or
andR-phycoerythrin[PE]-labeledanti-CD38(BDIS).Aliquotsof
two days before cell sorting, allogeneic BM stroma was irradiated
cells used for isotype controls were incubated for 30 minutes in 50
with 20 Gy and plated (7 X IO7 cells/well) in 96-well plates (Falcon:
pL of FITC-murine IgG (diluted 1:lOO: Coulter, Hialeah. FL) and
BD Labware. Lincoln Park. NJ) to form preestablished stromallayers
5 0 p Lof PE-murine IgG (diluted 150). After incubation. cells were
for the long-term cultures. Two hundred to five hundred cells with
washed once in phosphate-buffered saline (PBS). All FACS analysis
either the CD34-CD.78- or a CD34-CD38- immunophenotype were
and cell sorting was performed on a FACSVantage (BDIS) equipped sorted into each individual well of the 96-well plates and cultured
with an argon laser tuned to 488 nm and PCLysis I I and Cell quest
on the irradiated stromain "basal medium" containing IMDM, 308
software (BDIS). Figure IA represents forward and side scatter proFCS. 10% bovine serum albumin (BSA; Sigma), 2-mercaptoethanol,
file of the CD34' enriched population. RI defines the lymphoid gate
1O-6 m o l L hydrocortisone, penicillinlstreptomycin. glutamine. and
based onforward(size)andside(density)scatter.
CD34'CDW
various combinations of the growth factors: IL-3,
10 ng/mL: IL-6.
as thosecells in RI with high CD34
cellsweredefinedstrictly
50 U h L ; SF. 50 nglmL: and FL 50 U/mL.
antigen expression and anti-CD38 PE fluorescence less than half of
To measure progenitor content, adherent and nonadherent cells
themaximum PE fluorescenceoftheisotypecontrol.
In Fig IB,
were removed every 2 to 3 weeks, counted. and aliquots were reregion R2 represents the CD34'CD38- cells and region R3 defines
plated into semisolid media (1.3% methylcellulose with IL-3, I O ng/
the CD34TD38' cells.
mL; IL-6, 50 UlmL; SF, 50 ng/mL; GM-CSF. 50 ng/mL: and 2 U/
Flt3 ligand. The mouse and human ligands for FIt3 are structurmL erythropoietin. CFU-C and HPP-CFC were counted after 2 to 3
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Flt3 LIGAND'S EFFECTS ON CD34+CD38- CELLS
3565
medium with either ( I ) no growth factors, (2) FL alone. (3) 3/6/S.
or (4) F/3/6/S. Cultures were fed once a week by replacing half the
supernatant with fresh medium. Plates were observed every week
and wells with greater than 100 cells were considered positive. The
percent cloning efficiency was defined as the number of positive
wells divided by the total number of wells plated multiplied by 100.
Table 1. Effect of Adding FL to IL-3, 11-6, and SF on Early Cell
Proliferation From BM CD34' CD38- Cells Cocultivated 2 Stroma
Fold Increase in
Total Cells
Ratio Cell Numbers
Exp. No.
Stroma
3/6W
F/3/6/S
F/3/6/S:3/6/S
1
+
27
0
120
0
67
0
120
54
40
60
645
150
1,440
400
2,186
13
1,800
430
520
276
23.9
>150
12.0
>400
32.6
>13
15.0
8.0
13.0
4.6
15.6 z 3.6
-
+
2
-
+
3
-
+
4
5
6
7
Mean
2
+
+
+
SEM*
RESULTS
Effects of FL on skort-term proliferation from CD34CD38- cells. Our previous studies have shown that the
combination of IL-3, IL-6, and S F (3161s) induces cell proliferation from CD34TD38- BM and cord blood cells when
cocultivated on BM stroma." Our initial investigations into
the effects of FL determined whether the addition of FL to
the previously used combination of 3161s would enhance
short-term proliferation of CD34'CD38cells. BM
C D 3 4 T D 3 8 - cells were FACS sorted and cultured in either
3161s or F/3/6/S, with or without stroma. In both the absence
and presence of stroma, the addition of FL to 3/6/S significantly increased cell proliferation from BM CD34'CD38cells (Table I). In the absence of stroma, cell proliferation
from C D 3 4 T D 3 8 - BM cells grown in 3161s was not measurable. In contrast, when FL was added to the 3/6/S combination, cell number increased from day 0 to day 2 1 by 4- to
400-fold (n = 3). The presence of stroma significantly increased cell numbers in both 3161s and F/3/6/S, but the latter
combination still induced a higher proliferation than did 3/6/S.
Significant differences in cell cycling, cytokine responsiveness, and progenitor content have been previously noted
between CD34'CD38' cells and CD34TD38- cells in cord
blood and BM." We next compared the cytokine responsiveness of FL on these four functionally distinct progenitor
subpopulations (Fig 2). The most marked effect of FL was
seen on the most quiescent subpopulation of cells, the
CD34TD38- cells in BM, with a mean 15.6-fold greater
increase in cell expansion at day 21 in F/3/6/S than in 3/61
S (Table I ) (n = 7).
The effect of FL on cord blood CD34'CD38- cells was
less dramatic, with only a twofold greater expansion in F/3/
6/S than in 3161s (Fig 2). The smaller fold increase may
have been due in part to the marked proliferation of cord
blood cells in 3161s without the presence of FL. The effect
of FL on the more committed and cycling CD34'CD38'
Mean zS E M was calculated from all seven experiments including
only data from cultures containing stroma.
weeks in culture. HPP-CFC were defined as colonies with a diameter
greater than I mm, containing greater than S0.000 cells with a central
dense area surrounded by a 'halo of cells.' The cells not plated into
semisolid media were all replated onto irradiated stroma to continue
long-term culture. Long-term cultures were fed twice a week by
replacement of half the media.
FACS analysis of cultured cells. To analyze CD34' cell content
of cultures after stimulation in different growth factors, adherent and
nonadherent cells were removed after 21 to 30 days, counted,
washed, and incubated with 20 pL of human intravenous Ig (IVIg)
(10 mg/mL) for IO minutes to block nonspecific binding to Fc receptors. The cells were then stained with FITC-labeled anti-CD34 for
20 minutes. Cells from the same cultures were also stained with
isotype controls as described above. Approximately 30,000 events
were acquired in list mode data file and analyzed by Cellquest software. CD34' cells were defined as those in the lymphoid gate with
FITC expression greater than the isotype control. Cells showing a
nonspecific pattern of staining were excluded from the estimation
of CD34' cell frequency.
Cloning efficiency of CD34'CD3R- cells. To determine whether
FL increases the cloning efficiency of CD34'CD38- cells, BM
CD34'CD38- cells were sorted as single cells into 96-well plates,
using the ACDU (Automated Cell Deposition Unit) on the FACSVantage. The culture plates were prepared I to 2 days before cell
sorting with preestablished irradiated BM stromal layers and basal
Cord blood
Fig 2. Effect of FL on early cell expansion from
CD34+ cell populations in cord blood and BM.
CD34TD38' cells and CD34TD38- cells were sorted
onto a preestablished irradiated BM stromal layer at
500 cellslwell in a 96-well plate. Cells were cultured
on stroma in various growth factor combinationsof
(1) basal medium alone, (2) FL, (31 IL-3, IL-6, and SF
l3/6/S), and (41 F/3/6/S (see Materials and Methods).
Adherent and nonadherentcells were removed from
each culture and counted at day 21. The fold increase
of total cell output over input number is shown from
one representative experiment (of total seven BM
and t w o cord blood experiments).
Bone marrow
1
_
1
:
2 m -
fold Increase
cellnumber
Day 21
.
1oOO-
loo0
0
CD?d+CD38+
CD34+CD3%
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SHAH ET AL
3566
0
3
F13
m
6
F16
ea
m s
FIS
F1316
F13lS
F16lS
69
F1316lS
3.6,s
cells in cord blood and BM was significantly less than on
CD34TD38- cells from either source with no synergism
and less than additive enhancement when FL was added to
3161s. Thus, the synergism of FL with 3161s was restricted
to highly quiescent CD34TD38- cells of BM, a population
that is known to be relatively nonresponsive to other growth
factors in terms of delayed onset of proliferation and low
cloning efficiency."
Similar short-term proliferation studies were used to determine which of the individual cytokines in the 3/66 combination are synergistic with FL in inducing early proliferation
of BM CD34TD38- cells. FL was more effective as a single
agent than either IL-3, IL-6, or SF individually (Fig 3) (n =
3). IL-3 and IL-6 each induced early proliferation of
CD34TD38- cells in synergism with FL. The most impressive proliferation was seen with all four growth factors in
combination (F/3/6/S).
Cell proliferation from CD34'CD38- cells in long-term
culture with FL. Although synergism of FL with 3161s was
seen in both the presence and absence of stroma, culturing
A
-
4000
3000
Fig 3. Day 21 cell expansion from BM
CD34TD38- cells grown in multiple growth factor
combinations. BM CD34XD38- cells were sorted
onto a preestablished irradiated BM stromal layer at
500 cellslwell in a 96-well plate. Cells were cultured
in various growth factor combinations with and
without FL. Adherent and nonadherent cells were
removed from culture and counted at day 21.
cells without stroma for more than 21 days led to rapid cell
differentiation and a decrease in cell numbers in both 3161s
and F/3/6/S. To study the effect of FL in longer-term assays,
we chose to use stromal cocultivation for the longer culture
periods.
CD34TD38- cells were cocultured with stroma in various growth factor combinations for over 100 days. Total
adherent and nonadherent cells from each culture were
counted every 2 to 3 weeks. In combinations that contained
FL, cell expansion was greater at all time points and continued for longer than in the corresponding combinations without FL. Figure 4 shows the fold increase over time of cells
generated from BM CD34TD38- cells cultured on stroma
in various growth factor combinations in one representative
experiment of a total four experiments. In the cultures that
contained FL, there was an early peak of growth between
days 45 and 60 and a second smaller peak that appeared
around days 85 through 95. In contrast, cultures that did not
contain FL did not have the second peak of proliferation.
Although there is sample to sample variation as to the exact
0
-
-
3/s
F/6
6/S
-0-
3/6/S
F/3/6/S
3/6/S
--t F/3/6/S
F/S
Fold
Increase
2000
1000
0
0
20
40
60
Day oi long term cullwe
80
100
120
20
40
60
80
100
120
Day of longterm culture
Fig 4. Time course of cell expansion from BM CD34+CD38- cells. CD34TD38- cells were culturedfor over 100 days on stroma in the growth
factor combinationsshown. Every 2 to 3 weeks nonadherent and adherent cells were removed from each culture, counted, and returned to
the culture. The fold increase over input cell number is shown at each time point of long-term culture.
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3567
Flt3 LIGANDS EFFECTS ON CD34+CD38- CELLS
A
F13161S
Fl6IS
F131S
F1316
FIS
F16
F13
R
31615
615
31s
316
sa
IL6
IW
Colonies per 1 x 104 MIS
I
B
m
I C R K :
F1316IS
FI6IS
F131S
F1316
FIS
F16
F13
R
3161s
61s
Fig 5. The effect of FL on early and late progenitor
output. E M CD34TD38- cells were cultured on
stroma in the growth factor combinationsshown for
over 100 days. Adherent and nonadherentcells were
pooled and aliquots plated into methylcellulosemedium (see Materials and Methods) to measure the
frequency of progenitors (CFU and HPP-CFC) at (A)
day 30 and (B) day 56 time points.
31s
316
sa
IL6
IL3
0
timepoint of these two waves of cell proliferation, all experiments showed the same phenomenon.
Progenitor output from CD34'CD38- cells in long-term
culture with FL. To measure the progenitor content of the
cell expansion occurring in each growth factor combination,
adherent and nonadherent cells were pooled and aliquots
plated in methylcellulose every 2 to 3 weeks throughout
the duration of stromal cocultivation. Figure 5 shows the
frequency of CFU and HPP-CFC from the stromal cultures
at days 30 and 56 in one representative experiment. At day
30, CD34TD38- cells grown in all of the growth'factor
combinations were able to produce CFU. FL increased the
frequency of CFU when added to either IL-3 or IL-6 to the
same extent as did SF. However, HPP-CFC were seen at a
higher frequency in combinations containing FL. In contrast,
at day 56 all cultures containing FL produced a significantly
higher frequency of HPP-CFC and CFU than the corresponding growth factor combinations without FL ( P < .0025,
using square root transformation of the data). The addition
of FL to 3161s increased the frequency of CFU 20-fold at
10
20
30
40
50
60
Colonies per 1 x 104 Cells
day 56. At day 72, only combinations that contained FL
were able to produce either CFU or HPP-CFC (data not
shown).
CD34' cell content of cultures is maintained by FL. BM
CD34TD38- cells were sorted onto irradiated BM stroma
in either 3161s or F/3/6/S and analyzed by FACS for the
presence of CD34' cells after 21 to 30 days of culture (Table
2). In the presence of 3/6/S, 0.21 t 0.29%(mean -t SD, n
= 3) of the cultured cells retained CD34 antigen expression.
In contrast, 0.78% t 0.52% of the cells in cultures with Fl
3161.5 were CD34' after 21 to 30 days in culture. Taking
into account the greater overall cell expansion in Fl3/6/S,
the total number of CD34' cells in F/3/6/S was 20 to 126
greater than in 3161s.
FL increases cloning efJiciency of BM CD34'CD38- cells.
We next determined whether the marked proliferation of
CD34TD38- cells seen with FL is caused by recruitment
of normally cytokine nonresponsive cells to enter cell cycle
and proliferate. BM CD34TD38- cells were sorted as single
cells into 96-well plates onto preestablished stromal layers
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SHAH ET AL
b0Ml
n
3161s
F13I6lS
21
28
35
42
49
56
70
63
77
84
Day of Longterm Culture
with either no growth factors, FL, 3/6/S, or F/3/6/S. Cloning
efficiency was defined as the number of positive wells (wells
which contained at least 50 cells) divided by the total number
of wells plated. Wells were scored every 7 days to determine
the time of onset of clonal proliferation of each individual
CD34TD38- cell (Fig 6). The addition of FL to 3161s significantly increased cloning efficiency from a mean of 0.5%
with 3161s to 11.7% with F/3/6/S (P < .OOOl, using a Student's r-test).
We have previously shown in similar cloning efficiency
studies, that a subpopulation of CD34TD38- cells from
cord blood begin to proliferate as late as 80 days of culture
with 3I6lS.I' In the current studies with BM, a very low
cloning efficiency from CD34TD38- cells grown in 3161s
was seen with most clones appearing by day 35. Day 35
cloning efficiency was significantly higher in F/3/6/S (4.7%
u 0.26% in 3/6/S, P < .Owl).The addition of FL also
increased the percentage of CD34TD38- cells proliferating
late in culture (7.0% of cells in F/3/6/S u 0.24% in 3161s
first formed clones after day 35 (P < .OOOl). An analysis
Table 2. Effect of FL on Persistence of EM CD34+ Cells in Culture
% CD34' Cells
Exp. No.
1
2
3
Mean
SD
Day of
Analysis
30
23
21
Total No. of
CD34- Cells"
316s
FLY66
3/6/S
F/3/6/S
0
0.1
0.54
0.21
0.29
0.34
0.64
1.35
0.78
0.52
0
137
70
1,360
2,752
8,856
Total cell number of CD34' cells was calculated using the percentage of total mononuclear cells that remained CD34' (determined by
FACS analysis of the cultured cells) and the total number of mononuclear cells counted from each culture.
Fig 6. The effect of FL on cloning efficiency of EM
CD34TD38- cells. EM CD34TD38- cells were sorted
as single cells per well and cultured on stroma in
either basal medium, FL alone, 3/6/S, or F/3/6/S.
Plates were observed every week and wells with
greater than 100 cells were considered positive. Cumulative cloning efficiency over 84 days is shown
from two independent experiments.
of covariance of the data at all time points showed that F1
3/66 significantly increased the cloning efficiency of 3161s
over the entire 84-day period of culture (P = 0, power = 1,
using a Tukey's pairwise comparison procedure). Thus, the
addition of FL to 3161s significantly increased the frequency
of CD34TD38- cells able to proliferate at both early and
late time points. FL alone also resulted in a higher cloning
efficiency of CD34TD38- cells than did the 3161s combination with its predominant effect late in culture.
DISCUSSION
These studies show that FL is able to induce profound
cell proliferation from the primitive and relatively cytokine
nonresponsive CD34'CD38- progenitor population in BM.
Single-cell plating shows that the cell output induced by FL
is due at least in part to its ability to increase the percentage
of BM CD34'CD38- cells able to respond to the cytokines
IL-3, IL-6 and SF. Evidence that FL has unique effects on
the most functionally primitive progenitors is provided by
its dominant effect on progenitor output late in culture.
In this report, both the synergism of FL with other growth
factors and its action as a single agent were significantly
more impressive than in previous studies of both human and
murine ~pc.6.7.IO.l3The proliferative effects of FL do not
appear to overlap entirely with SF as previously suggested.7.9.IO.13 In our studies, the effects of FL were most
profound in the most quiescent subpopulation, the BM
CD34TD38- cells, and were minimal in the more mature
and cytokine responsive CD34TD38' cells. Cord blood
CD34TD38- cells are more responsive to IL-3, IL-6, and
SF than are BM CD34'CD38- cells with a more rapid onset
of proliferation and higher cloning efficiency." The effect
of FL on cord blood CD34TD38- cells was accordingly
less impressive than on CD34'CD38- cells in BM. The use
of human progenitor populations which are less quiescent
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Flt3 LIGANDS EFFECTS ON CD34+CD38- CELLS
3569
been disappointingly low in all clinical trials reported thus
and respond more readily to cytokine stimulation will not
far, presumably because of the inability to induce cell cycling
show the unique effect of FL on quiescent cells demonstrated
of true stem cells during a short transduction p e r i ~ d . ' ~ - ' ~
in this r e p ~ r t . ' ~ . ' ~ . ' ~
Prolonged cytokine stimulation in vitro leads inexorably to
Previous studies of FL have measured proliferation and
mature and primitive HPC. The studies
progenitor output in relatively short-term a s ~ a y s . 6 ~Th~e ~ ~ ~the~ ~loss
' ~ of
~ ' both
~
reported here show that FL is able to induce proliferation
effects of FL on progenitor output in short-term culture of
of primitive and normally cytokine resistant hematopoietic
cytokine responsive cells are similar to those of SF, revealing
progenitors and may enhance maintenance of HPC in vitro,
a degree of redundancy between the two cytokines. In our
suggesting that stimulation with FL may provide a significant
own studies the unique effect of FL on progenitor output
improvement in gene transfer into human hematopoietic
was not apparent until the end of the standard LTC-IC period
stem cells.
(day 56) and was most impressive in the ELTC-IC period
(day 72). Thus, FL acts on a subpopulation of CD34+CD38cells that produce progenitors late in culture, likely representACKNOWLEDGMENT
ing a more primitive subpopulation of HPC.
The
authors
thank
Geralyn
Annett and Flavia Thiemann for their
Two other studies have used stromal long-term cultures
invaluable technical assistance and Earl Leonard for biostatical analto show the ability of FL to induce proliferation of primitive
ysis. We also thank Dr Robertson Parkman and Dr Donald B. Kohn
progenitors. Small et a14 found that antisense oligonucleofor advice on preparation of the manuscript. We also give special
tides against STK-1 (the human homolog of murine Flt3)
thanks to the Labor and Delivery staff at Kaiser Permenante Sunset,
inhibited production of CFU most strongly in "long term"
Los Angeles for their generous assistance in providing umbilical
(3 to 4 week) stromal cocultures. Gabbianelli et all4 recently
cord blood for use in these studies.
reported that FL increased progenitor output in 5 to 7-week
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1996 87: 3563-3570
Flt3 ligand induces proliferation of quiescent human bone marrow
CD34+CD38- cells and maintains progenitor cells in vitro
AJ Shah, EM Smogorzewska, C Hannum and GM Crooks
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