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A Murine Stromal Cell Line Allows the Proliferation of Very Primitive Human
CD34 + -I-/CD38 - Progenitor C e l l s in Long-Term Cultures
and Semisolid A s s a y s
By Cherifa Issaad, Laure Croisille, Andre Katz, William Vainchenker, and Laure Coulombel
Analysis of molecular mechanisms associated with stem
cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem
cells in human bone marrow. Such primitive stem cells
usually do not form colonies in short-term semisolid assays
and are best identified by their ability to initiate sustained
hematopoiesis when they are cocultured with competent
stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5)that supports colonyforming unit-spleen (CFU-S) maintenance would permit,
both in short-term colony assays and long-term cultures,
the development of primitive human stem cells sorted on
the basis of their high expression of CD34 and lack of
expression of CD38 antigen. In short-term colony assays,
this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination
of interleukin-3, Steel factor, granulocyte colony-stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning
efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development
of multipotent blast cell progenitors with a high proliferative
potential, which in these conditions represented 1% to 2%
of CD34+ +/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much
lower numbers of CFU-blast were detected. This selective
action of MS-5 on early progenitorswas also observed when
MS-5 cells were used as feeders in long-term cultures of
CD34+ +/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells
up t o 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial
CD34+ +/CD38- cells as estimated.by limiting dilution experiments. Mechanisms by which murine stromal cells act
specifically on human primitive stem cells are unclear, but
from our data this effect is unlikely t o be explained solely
by known species cross-reactive growth factors. Further
manipulation of this long-term coculture system should
prove useful in identifying stromal molecules regulating
commitment and differentiation of early human progenitor
cells.
0 1993 by The American Society of Hematology.
M
several weeks in the cocultures must represent the progeny
of more primitive progenitor cells referred to as long-term
culture-initiating cells (LTC-ICS)~." present in the input
population. The successful use in transplantation of human
cells maintained in long-term cultures" as well as the in vitro
expansion of totipotent murine hematopoietic stem cells with
myeloid and lymphoid repopulating p ~ t e n t i a l 'show
~
that
very primitive multipotential stem cells are maintained in
these conditions. Moreover, recent data suggest that these
long-term cultures might reproduce in vitro many regulatory
aspects of the in vivo situation and might permit investigators
to separately assess differentiation (production of clonogenic
cells) and maintenance (indicative of some self-renewal) of
LTC-ICS.'~~'~
Long-term cultures were initially established with unfractionated marrow samples that provided both the stromal cells
and the hematopoietic stem cell^.^.^ Subsequently, in vitro
systems have been simplified by adding marrow cell suspensions enriched in CD34+/CD33- or CD34+/HLADr- primitive progenitor cells to pre-established irradiated, confluent,
allogeneic adherent layers derived from 4- to 6-week-old
classical long-term culture^.^^^^'^ Until recently, it has been
assumed that only marrow stromal cells of human origin
would be able to support the development of early human
progenitor cells because most of human growth factors known
to be necessary at these early stages (ie, interleukin-3 [IL-3],
IL-6, and granulocyte-macrophage colony-stimulating factor
[GM-CSF]) are not species cro~s-reactive.'~~'~
However,
studies from several laboratories have reported that a xenogenic environment was permissive in vivo and, although to
a lesser extent, in vitro for human pluripotent stem cell development. Indeed, some murine stromal cell lines appear
to be as effective as human feeders in supporting the mainte-
OST OF OUR knowledge on the fundamental properties of hematopoietic stem cells relies on in vivo
studies performed in the murine model in which a hierarchy
of transplantable stem cells with different self-renewal and
differentiation potentials has been
Evidence that
such stem cells exist in humans is only indirect and comes
from the observation that multiple lineages are derived from
neoplastic clones4and from examples of clonal hematopoietic
reconstitution after marrow tran~plantation.~
The most reliable in vitro experimental approach to study the biology of
these very primitive human stem cells consists of growing
human marrow cells for periods of up to 8 to 10 weeks in
the presence of a competent underlayer of marrow stromal
Under these conditions, it is assumed that colonyforming cells present at the initiation of the culture either
differentiateor die. Therefore, clonogenic cells recovered after
From INSER UM U 362, Institut Gustave Roussy, Villejuif;France.
Submitted September 22, 1992; accepted January 22, 1993.
Supported by INSERM and by grants @om Association pour la
recherche contre le cancer, Lime nationale contre le cancer, Fondation
de France, and Fondation Jacques et Monique Roboh and Suzanne
Axel, and CNAMTS.
Presented in part at the XXXIII Meeting of the American Society
of Hematology, Denver, December 6-10, 1991, and published in abstractform (Blood ?8:301a, 1991 [supplI ] ) .
Address reprint requests to Laure Coulombel, MD, PhD, INSERUM U 362, Institut Gustave Roussy, 94805 Villejuif;France.
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 1993 by The American Society of Hematology.
0006-49?1/93/811 I -0030$3.00/0
2916
Blood, Vol81, No 1 1 (June l ) , 1993: pp 291 6-2924
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291 7
MURINE STROMAL CELLS SUPPORT HUMAN STEM CELLS
nance and the myeloid and lymphoid differentiation of unfractionated hematopoietic cells,I8 CD34++/HLADpW,’4or
Selection of Cells by Cell Sorting
Cells were analyzed and sorted on an ODAM ATC 3000 cell sorter
CD34+/Thy 1
pluripotent stem cells. H u m a n multipotent
(ODAM/Bruker, Wissembourg, France) equipped with an INNOVA
cells can also be successfully engrafted in vivo into fetal
70-4 Argon ion laser (Coherent Radiation, Palo Alto, CA) tuned at
sheepz0r2’ or into genetically immunodeficient (SCID)
488 nm and operating at 500 mW. The wide angle light scatter
mice.22~23
However, in most xenogenic settings, the prolif(WALS) was first extracted with a 5 I O DCLP filter. The two fluoeration and terminal differentiation of grafted human cells
rescence signals were then separated with a 550 DCSP filter and finally
remain limited unless human growth factors are p r ~ v i d e d . ’ ~ , ~ ~isolated using a 530 DF 30 filter for FITC and a 575 DF 26 filter for
PE (all filters from Omega Optical Inc, Brattelboro, VT). CompenIn this study, we attempted to selectively maintain and
sation for double-stained samples was set up with single-stained samexpand very primitive human progenitor cells that could be
ples. Data were analyzed on the ATC 3000 computer and all paramsubsequently used for the molecular analysis of the mechaeters were displayed on a linear scale. Three sorting gates were set to
nisms associated with their developmental potential. To that
include cells expressing both CD38 and CD34 antigens (referred to
purpose, we constructed a long-term culture assay by coculas CD34+/CD38+ cells), cells “negative” with respect to CD38 and
turing primitive h u m a n marrow cells selected for their high
expressing high levels of CD34 (the 30% CD34 most positive among
expression of the CD34 antigen and very low expression of
the CD38 negative/CD34 positive cells referred to as CD34’+/CD38CD38” with a murine clonal stromal cell line (MS-5). MScells), and the remaining CD34+/CD38- cells with medium expression
5 was derived from noninfected Dexter-type murine longof the CD34 antigen. To increase the purity of the sorting, the morterm marrow cultures and supports colony-forming unitphologic two-parameter histograms (WALS v electric measurement
of the cell volume) were acquired for the cells falling in each of the
spleen (CFU-S) maintenan~e.’~.’~
Our results indicate that
three previous gates, and an additional gate was applied to each of
MS-5 cells, but not h u m a n feeders, allow the expansion of a
these morphologic distributions to retain a well-defined homogeneous
subpopulation of very early h u m a n multipotent progenitor
population and to reject highly diffusive cells (which may be autocells for 5 to 10 weeks without the need for exogenously
fluorescent) and/or too large objects (which may correspond to cell
supplied growth factors. Furthermore, we show that MS-5
doublets). The four-parameter sorting was run at 3,000 to 3,500 cells
cells act synergistically with human growth factors to stimper second and the sorted cells were collected in 3.5 mL of aMEM
ulate the formation of blast colonies and macroscopic colonies
with 10% FCS.
from high proliferative potential multipotent CD34++/CD38cells i n short-term methylcellulose assays.
MATERIALS AND METHODS
Marrow Cell Preparation
Bone fragments were obtained after informed consent from patients
undergoing hip surgery, and marrow cells were collected by vigorous
shaking of bone fragments in (Y minimal essential medium (aMEM)
supplemented with 100 pg/mL of deoxyribonuclease(Sigma Chemical
Co, St Louis, MO; DNase type I). Cells were centrifuged once,
counted, and separated on Ficoll-Hypaque. Light-density ( < I .077 g/
mL) cells were suspended at a concentration of 1 X lo7 cells/mL in
aMEM supplemented with 30% fetal calf serum (FCS; JBio Laboratory, Les Ulis, France) and kept at 4°C overnight. The next day,
the cells were carefully resuspended in fresh (YMEMwith 5% FCS
and 100 fig/mL DNAs (labeling medium) and proceeded for labeling.
Cell Separation
Cells (2 X lo7 cells/mL) were first incubated with the anti-CD34
monoclonal antibody (MoAb) 8G12 (kindly provided by P. Lansdorp,
Terry Fox Laboratory, Vancouver, Canada, as purified Ig and used
at a concentration of 14 pg/mL), followed by 45 minutes of incubation
with a 1/30 dilution of phycoerythrin (PE)-labeled antimouse IgGl
goat Ig (Southern Biotechnology, Birmingham, AL). Remaining free
sites on the PE-Ig were blocked by 15 minutes of incubation with
mouse serum (1/5 dilution). Cells were subsequently labeled with
anti-CD38 MoAb directly coupled with fluorescein (Immunotech,
Marseille, France) and used at a 1/10 dilution. Cells were finally
suspended in labeling medium at a concentration of 4 to 5 X IO6
cells/mL and separated by cell sorting. More recently, cell labeling
has been performed in one step by incubating cells simultaneously
with the MoAb HPCA-2 (8G12) directly coupled to PE (Becton
Dickinson, San Jose, CA) and the CD38-fluorescein isothiocyanate
(FITC) MoAb.
Assessment of Clonogenic Progenitors in the Different
CD34/CD38 Cell Fractions
Erythroid (CFU-erythroid [CFU-E] mature burst-forming uniterythroid [mBFU-E] and immature BFU-E (iBFU-E]), granulocytic
(CFU-granulocyte-macrophage[CFU-GM]), and (CFU-granulocyteerythroid-monocyte-megakaryocyte[CFU-GEMM]) progenitors were
quantified using previously described methylcellulose assays.6.*’ Each
of the three fractions selected as described above (CD34++/CD38-,
CD34+/CD38-, and CD34+/CD38+)were plated at a concentration
of 500 to 1,000 cells/mL of complete methylcellulose medium (0.8%
methylcellulose in Iscove’s medium, 30% FCS, 1% deionized bovine
serum albumin [BSA], I 0-4 mol/L 0-mercaptoethanol). Colonystimulating factors were provided either as 10% of agar-leukocyte
conditioned medium (A-LCM [an usual source of hematopoietic
growth factors]) or as recombinant growth factors: rhuSteel factor
( S F kindly provided by Amgem, Thousand Oaks, CA), recombinant
human IL-3 (rhuIL-3), recombinant human granulocyte-CSF(rhuGCSF), and rhuIL-6 (all purchased from Genzyme, Cambridge, MA)
used at the concentrations indicated in Results. Human erythropoietin
(Epo; purified from human urine and purchased from the Terry Fox
Laboratory) was added to every growth factor combination at 3 U/
mL. Plates were incubated at 37°C in an air atmosphere supplemented
with 5% C 0 2 and saturated with humidity. Progenitors were scored
at day I O (CFU-E and mBFU-E), days 15 and 16 (primitive iBFUE and CFU-GM), and days 22 to 24 (CFU-GEMM and blast progenitors) using previously detailed
Parallel experiments were performed to test the ability of different
stromal cells populations to alter the clonogenic potential of sorted
CD34++/CD38- cells. In these experiments, 10,000cells ofthe murine
MS-5 cell line or 50,000 irradiated human stromal cells were added
per milliliter of the methylcellulose mixture and cells were plated in
bacteriologic plastic dishes (Falcon 1008) that allow spreading of
stromal cells but slow their proliferation as compared with plating
on tissue culture dishes.
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2918
ISSAAD ET AL
Table 1. Hematopoietic Progenitor Cell Content of the Different Fractions Sorted According to the Expression
of Both CD34 and CD38 Antigens
~
Cell Fraction and
Growth Factors
CD34++/CD38- (n = 9)
SF IL-3 Epo
A-LCM EPO
CD34+/CD38- (n = 5)
SF + IL-3 + EPO
A-LCM t EPO
CD34+/CD38+ (n = 3)
SF IL-3 + EPO
A-LCM EPO
+
+
+
+
+
~
~
~
~
~~
Progenitor Cells per 1.000 Cells Plated (mean r SEM)
CFU-E
mBFU-E
IBFU-E
CFU-GEMM
CFU-GM
CFU-Blast
0.9 f 0.3
0.5 t 0.1
6 ? 1.6
1.7 f 0.6
5.3 2 1
0.6 % 0.2
0.8 f 0.5
ND
17+4
15f4
1 k 0.3
ND
1054
9*5
41 ? 10
26 f 8
16%3
521
3 t 1.2
0.7 t 0.3
25 k 5
41 k 8
84 f 48
139 k 13
91 f 2 3
5 4 f 12
ND
ND
66 f 7
126 f 35
4 t 0.3
3 2 0.7
1.6f 1
ND
ND
ND
One thousand cells from each sorted CD34/CD38 fraction were plated in methylcellulose assays in the presence of either 10% A-LCM or a mixture
of rhuSF (50 ng/mL) and rhulL-3 (100 U/mL). Epo (3 Ul/mL) was added in each dish. Colony counts were performed in duplicate or triplicate between
days 12 and 22. Each number refers to the mean (fSEM) number of progenitor of each type detected in three to nine experiments.
Abbreviation: ND, not detected.
Long-Term Cocultures of CD34++/CD38- Cells With
Various Stromal Cell Lines
Selection ojStromal Cells
Human stromal cells. Human marrow stromal cells were derived
from 4- to 6-week-old human long-term marrow cultures established
as described previously? At least 72 hours before their use as feeders
in cocultures, long-term marrow culture flasks or dishes were irradiated at 10 Gy and the adherent layer was replated 48 hours later
in 24-well plates (6 to 8 X 1O4 cells/well in 1 mL) in long-term culture
medium (LTC-medium; aMEM supplemented with 12.5% horse
serum [Hyclone Laboratories, Logan, UT], 12.5% FCS,
mol/L
hydrocortisone, and
mol/L 2 P-mercaptoethanol).
Murine stromal cells. The MS-5 stromal cell line was kindly provided by K. MoriZ6and maintained in aMEM supplemented with
10% FCS. The cell line is contact inhibited and can be kept at confluence for several weeks without needing to be replated. For coculture
experiments with enriched human hematopoietic progenitor cells,
unirradiated MS-5 feeders were pre-established in 24-well (4 X lo4
cells/well in 1 mL) or in 96-well plates (6 X IO3 cells/well in 100 pL).
To avoid early detachment of the stromal layer, plastic wells were
precoated with 1% gelatin immediately before seeding of the stromal
cells.
Establishment of Cocult ures
Cocultures were established by incubating cells of the three sorted
fractions (CD34++/CD38-, CD34+/CD38-, or CD34+/CD38+cells)
either in 24-well plates (2,000 to 5,000 cells/well) or in 96-well plates
(2 to 100 cells/well) precoated with human or murine stromal cells.
Cultures were initiated in LTC-medium and fed every week by halfmedium change. Cocultures were sacrificed at regular intervals and
the progenitor content of each well was assayed in methylcellulose
colony assays. Cocultures initiated in 24-well plates were sacrificed
at least once after 4 or 5 weeks in culture. Progenitor content was
again assessed after 7 weeks if a sufficient number of purified CD34++/
CD38- cells was available to initiate multiple wells. Nonadherent
and adherent fractions (the latter detached by a short trypsinization
and plated at 3 to 4 X lo4 cells/mL) were separately assessed. In
contrast, for limiting dilutions, the entire content of each well of the
96-well plate was harvested and plated in 1 mL of methylcellulose
medium after trypsin treatment, and at least I O different wells were
terminated at each time point. Culture conditions of the colony assays
were as described earlier for the evaluation of the clonogenic potential
of the different sorted populations.
RESULTS
CD34++/CD38- Cells Are Enriched in Multipotent High
Proliferative Potential Primitive Progenitor Cells
The clonogenic progenitor content of the three sorted populations (CD34++/CD38-, CD34+/CD38-, and CD34+/
CD38+) was first assessed by plating 500 to 1,000 cells of
each population in methylcellulose assays. Maximal numbers
of each progenitor type found in each fraction are indicated
in Table 1. We first noticed that the level of expression of
the CD38 antigen discriminated functionally distinct progenitor cells among CD34' cells. The CD34+/CD38+ population included 25%to 30%clonogenic progenitors with a
mature phenotype, ie, CFU-E, mBFU-E, and small CFUGM generating colonies ofless than 200 cells. In the presence
of Epo, these cells could be stimulated by either rhuSF (50
to 100 ng/mL) or rhuIL-3 (50 to 100 U/mL). In contrast,
most of the primitive progenitors, iBFU-E, CFU-GEMM,
and high proliferative potential CFU-GM (generatingcolonies
of more than lo4 cells) were CD38- and expressed medium
to high levels of CD34+. The cloning efficiency of the CD34+/
CD38- fraction was 8% to 16% and that of the CD34++/
CD38- fraction was 2% to 3%(Table 1). Colony formation
by CD34++/CD38- cells did not occur when cultures contained only one cytokine or A-LCM in addition to Epo, but
required at least the combination of SF IL-3 in addition
to Epo, in agreement with the multiple cytokine requirement
of early stem cells previously reported.29
In contrast to the modest stimulatory effect of soluble recombinant growth factors on CD34++/CD38- cells, addition
of murine stromal MS-5 cells to the colony assay dramatically
increased the number and size of the colonies detected. MS5 cells act only in synergy with human growth factors and
did not stimulate colony formation by CD34++/CD38- cells
when added alone (some < I O cells clusters were observed in
the CD34+/CD38- or CD34+/CD38+ fractions). In five experiments, CD34++/CD38- cells were plated in the presence
of MS-5 and either A-LCM Epo (Fig IA) or SF IL-3
Epo (Fig 1B). In both conditions, the cloning efficiency increased threefold to fourfold over that observed in the pres-
+
+
+
+
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MURINE STROMAL CELLS SUPPORT HUMAN STEM CELLS
70
ci,
T
+
+
d
60
2919
A
A-LCM + EPO
0
T
50
m
8
.-ii
-a)
ci,
T
m
40
o
0
30
,- 30
z
Q
20
20
0
c
.-
c.
0
0
0
40
0-
Q
.-c
50
d
9
0
60
5 lo
10
OJ
iBFU-E
I CFU-GM
1
mBFU-E
CFU~GEMM
CFU-Blast
I
E
o
1
iBFU-E
I CFU-GM
CFU-GEMM
CFU-Blast
~
mBFU-E
Fig 1. Synergistic effect of MS-5 murine stromal cells and growth factors on the cloning efficiency of CD34+ +/CD38- cells. One
thousand CD34+ +/CD38- cells were plated in Iscove’s medium methylcellulose assays with different growth factor combinations: (A)
Epo (3 Ul/mL)
10%A-LCM; (E) Epo (3 Ul/mL)
IL-3 (100 U/mL)
SF (50 ng/mL). Cells were cultured either without (0)or with (m)
10,000 MS-5 cells. Each column represents the mean ( 2SEM) number of progenitors observed in five separate experiments. ‘P < .01
(paired analysis).
+
+
ence of growth factors without stromal cells, ie, 10%-t 1.3%
versus 2% -t 0.3% for A-LCM
Epo, and 11.2% -t 2.6%
versus 3.7% -t 0.8% for S F + IL-3 Epo with or without
MS-5, respectively (Fig IA and B). Differences expressed in
Fig 1A and B were statistically highly significant (P < .01,
paired analysis of data, Student’s r-test) for iBFU-E, CFUGEMM, CFU-GM, and CFU-blast (see below). Most erythroid and granulocytic colonies obtained in colony assays
stimulated by MS-5 cells included more than 2 X IO5 cells,
a macroscopic size that is much higher than that observed
+
+
Fig 2. Photographof a blast cell progenitor observed at day 22 in methylcellulose
colony assays of CD34+ +/CD38- cells cultured in the presence of A-LCM
Epo
10,000 MS-5 cells. This colony was individually replated and generated 97 secondary
8 BFU-E). (Original magcolonies (89 GM
nification X 200.)
+
+
+
in the absence of the stromal cells. This great cell proliferation
was predominantly seen in the erythroid lineage, which led
to an underestimation of the number of multilineage colonies.
In addition to BFU-E and CFU-GM, MS-5 in synergy with
A-LCM or S F IL-3 Epo triggered the development of a
very primitive progenitor, which shared some of the criteria
identifying blast cell progenitors (CFU-blast) (Fig 2). ( I ) These
colonies were undetectable before day 15 and were characterized by their small size (<300 cells) at days 22 and 23 (Fig
2), which contrasted with the macroscopic size of CFU-GM-
+
+
+
C
%
g
’
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2920
ISSAAD ET AL
Table 2. Replating Potential of Primary Blast Cell Colonies
Generated by CD34++/CD38- Cells
No. of
No. of Primary
Colonies
No. of Primary
Experiment
No.
Colonies
Repeated
Generating
Secondary
Colonies
3
4
5.
6
26
35
14
17
24 (94)
32 (91)
11 (78)
16(94)
Secondary
Colonies per
Primary Colony
(mean ? SEM)
Secondary
Plates With
Erythroid and
Granulocytic
Colonies
34 t 5
129 t 17
36 t 6
154t3
19 (73)
21 (60)
6 (54)
9 (53)
No. of
One thousand CD34++/CD38- cells were grown in methylcellulose
colonyassays with MS-5, Epo, and A-LCM. At day 22, colonies defined
as blast colonies (see text and Fig 2) were individually lifted and replated
in secondary methylcellulose plates containing Epo + A-LCM without
MS-5. Secondary colonies were scored at days 12 through 15. Percentages are in parentheses.
and BFW-E-derived colonies. (2) They were composed of
cells with an undifferentiated morphology after May-Grunwald Giemsa staining. (3) They generated high numbers of
secondary colonies of both erythroid and granulocytic lineages
after individual replating; thus, 83 of the 92 primary colonies
individually replated gave rise to secondary colonies (Table
2). Each primary colony generated on average 88 (range, 10
to more than 400) secondary colonies and 55 yielded both
erythroid and granulocytic colonies, indicating that the parental blast progenitor was pluripotent (Table 2). In 15 colonies in which it has been calculated, the replating efficiency
(number of progenitor cells per number of cells in the primary
colony) ranged from 10% to 100% (mean k SEM, 49% 2
9%).The frequency of these progenitor cells in the CD34++/
CD38- population was 1% (Fig IA and B). Irradiation of
MS-5 did not abrogate its supportive effect (data not shown).
In one experiment that used substitution of MS-5 by human
stromal cells derived from irradiated long-term marrow cultures, no progenitors of blast cell colonies were observed.
Such high proliferative potential progenitor cells were not
detected in the CD34+/CD38+population, but were present
in the CD34+/CD38- fraction, although their detection was
obscured by the high number of committed progenitor cells.
Long-Term Maintenance of Hematopoiesis
by CD34++/CD38- Cells Cocultured With Human
and Murine Stromal Cells
To determine whether cells able to initiate long-term hematopoiesis in vitro were present among CD34++/CD38-,
we cocultured CD34++/CD38- cells with stromal cells of
murine (MS-5) and human origin for 5 to 10 weeks in culture
conditions previously shown to support long-term myelopoiesis.6
Analysis of the Supportive Capacity of Murine Stromal
MS-5 Cells
In a first set of experiments, 5,000 CD34++/CD38- cells
sorted from 4 different marrow samples were incubated in
24-well plates precoated with unirradiated MS-5 murine
stromal cells and the progenitor content of the wells was determined at regular intervals during the 5- to 10-week coculture period. In each of these four experiments, the number
of clonogenic progenitor cells recovered from each culture
well increased 3- to IO-fold during the culture period (Fig 3).
Interestingly, this amplification was restricted to the compartment of primitive progenitors, ie, iBFU-E, high proliferative potential CFU-GM, and CFU-GEMM. Blast cell progenitors were also detected for several weeks, although there
was no clear increase in their numbers. Interestingly, the proportion of iBFU-E and CFU-GEMM detected in the cocultures was always greater than 30% of the total number of
progenitors, an observation that might be related to the selective enhancement of erythroid proliferation by MS-5
mentioned earlier. In contrast to its action on early progenitor
cells, murine stromal cells did not support terminal differentiation of human cells. This was indicated by two observations: (1) the very low number of mBFW-E and day 7 CFUGM produced, and (2) even though there was on average a
threefold to fourfold increase in the number of nonadherent
cells produced at week 5, terminally differentiated granulocytic cells were not observed.
Finally, in two experiments in which CD34++/CD38- cells
were cocultured 4 to 5 weeks simultaneously on MS-5 and
human stromal cells, we studied the distribution of progenitor
cells in the culture. In both experiments, more than 80% of
the progenitor cells were associated with adherent stromal
cells independently of their human or murine origin.
Analysis ofthe Proliferative Capacity of CD34++/CD38
Cultured at Limiting Dilutions on MS-5 Feeders
In an attempt to determine whether the production of
primitive progenitors observed in these cocultures resulted
3,500
=
a,
3,000
3
&
2,500
Q
v)
=
8
2,000
.E
1,500
a,
UI
1,000
c
2
n
0 1 2 3 4 5 6 7 8 910111213
Weeks in culture
Fig 3. Production of clonogenic progenitor cells from long-term
cocultures initiated with 5,000 CD34+ +/CD38- cells sorted from
4 different marrow samples and cultured in 24-well plates precoated
with a confluent layer of MS-5 cells. Wells were sacrificed at regular
intervals and their progenitor content quantitated in methylcellulose
colony assays. Each point represents the total number of progenitors
present in a 24-well culture (ie, per 5,000 initial CD34+ +/CD38cells) at the time point indicated. Input (day 0) progenitorvalues for
the four experiments were 1 8 5 (Exp 2). 5 5 (Exp 31, 365 (Exp 41,
and 500 (Exp 5).
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2921
MURINE STROMAL CELLS SUPPORT HUMAN STEM CELLS
one well) after 4 weeks of coculture on MS-5 favors the hypothesis that a fraction of very immature pluripotent progenitor cells is responsible for the sustained clonogenic output
observed in these culture conditions.
Analysis of the Supportive Capacity of Human Stromal
Cells
I .
1
. . . . . . .I
10
. . . . . . . .I
100
.
.
, , . . . .
I
,
1,000
. . . . .J
5,000
Initial CD34++/CD38- cells per well
Fig 4. Limiting dilution analysis of data from a representative
experiment in which decreasing numbers of CD34++/CD38- cells
were seeded in 96-well plates precoated with MS-5 cells. At weeks
5 and 7, microwells initiated with I O , 100, and 5,000 C D 3 4 + + /
CD38- cells were harvested and their progenitor content assessed
in methylcellulose colony assays. Note that MS-5 were therefore
present in each colony assay. Each point represents the mean
( ? SEM) number of progenitor cells per positive well (positive wells:
12 of 1 9 [week 5. 10 cells/well]. 7 of 23 [week 5, 10 cells/well],
1 8 of 1 8 wells [week 4, 100 cells/well], 10 of 10 wells [week 7,
100 cells/well]). The SEM is not indicatedfor the 5,000 cells point
as only one well was established at this cell concentration.
from the clonal expansion of a small number of very immature CD34++/CD38- pluripotent cells, we initiated cocultures at limiting dilutions by incubating from 2 to 100
CD34++/CD38- cells in 96-well plates precoated with MS5. We first examined the relationship between the clonogenic
output after 5 weeks and the number of CD34++/CD38- cells
initiating the coculture. As shown in Fig 4 for one representative experiment, the number of clonogenic cells detectable
after 5 and 7 weeks in culture was linearly related to the
number of initial cells. In each of five different experiments,
100% of the wells initiated with 50 or 100 CD34++/CD38cells and sacrificed at 4, 6, and 8 weeks (10 wells per time
point) were positive, ie, yielded at least one clonogenic progenitor cell. Eighty percent of wells initiated with 20 cells
and sacrificed in the same conditions as previously were also
positive, but only 65% of those containing 10 cells and 1 I%
of the I80 wells initiated with 2 cells and sacrificed between
weeks 4 and 10 (20 wells per time point). However, there
was a wide variation in the number of progenitor output
measured in individual wells at weeks 5 through 10. Wells
initiated with 100 cells yielded between 4 and 78 progenitor
cells. For wells initiated with 2 CD34++/CD38- cells, the
number of progenitors produced ranged from 1 to 29, respectively, with an average of 3.4 progenitor cells per positive
well. As the cloning efficiency of the starting CD34++/CD38population is at most 10%(Fig 1A and B), each well initiated
with 2 cells can be assumed to contain less than 0.2 progenitors at the initiation of the coculture on MS-5. Our finding
that 4 of the 180 wells initiated with 2 CD34++/CD38- cells
contained more than 4 primitive progenitor cells of both the
erythroid and granulocytic lineages (up to 29 progenitors in
In parallel experiments, CD34++/CD38- cells were incubated on 4-week-old irradiated human stromal layers following the same procedure as described for cocultures of CD34++/
CD38- on MS-5. As indicated in Table 3, the progenitor
output in cocultures on human stromas was always lower (4to 60-fold less) than that measured in cocultures on MS-5 at
any time point during the coculture period. The addition of
MS-5 cells to the colony assays of cells maintained on human
stromas failed to increase progenitor cell recovery, indicating
that the low progenitor output was not due to suboptimal
stimulatory conditions in the clonogenic assay.
Long-Term Cultures of CD34+/CD38+ Cells on MS-5
Cells and Human Stromal Cells
In two separate experiments, we assessed the clonogenic
output of CD34+/CD38+ cells cocultured with either MS-5
or human stromal cells. As shown in Table 3, cells from this
fraction were unable to maintain a high clonogenic output
when cocultured on either murine or human stromal cells,
a result that was not unexpected considering that the CD38+
fraction is very enriched in mature clonogenic progenitor
cells.
DISCUSSION
Although some primitive pluripotent stem cells will form
c o l ~ n i e sin' ~short-term
~ ~ ~ ~ ~colony
~
assays, their identification
is indirect and usually relies on the measurement of their
clonogenic progeny after 5 to 8 weeks of culture with stromal
cells. These long-term coculture systems do not require the
Table 3. Clonogenic Output in Long-Term Cultures of CD34++/
CD38- and CD34+/CD38+ Cells on Either Murine
or Human Stromal Cells
Progenitor Cells/Well
Weeks in
Culture
CD34++/CD38Exp 3
Exp 5
5
5
11
CD34+/CD38+
Exp 3
Exp 5
5
5
11
MS-5
Human
Stroma
808
1,912
1,392
200
189
21
649
22
0
128
4
0
Long-term cocultures were initiated by plating 5,000CD34++/CD38or CD34+/CD38+cells in 24-well plates precoated with confluent layers
of either MS-5 cells or irradiated human stromas. The progenitor content
of each well was assessed at weeks 5 and 11 by harvesting the adherent
and nonadherent fractions that were plated in methylcellulose assays.
Numbers refer to the total progenitor content of each well (adherent and
nonadherent fractions).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2922
ISSAAD ET AL
addition of exogenous growth factor^.^,^,^' Because of the lack
of species cross-reactivity of most known growth factors, syngeneic stromal cells have usually been preferred to xenogenic
feeders. However, recent evidence has accumulated showing
that human hematopoiesis can develop in a nonhuman envir~nment.~~
In' ~
this
~ -study,
' ~ we show that a murine stromal
cell line (MS-5), selected for its ability to support day-12 CFUS and long-term reconstituting stem cell maintenance (Sainteny et al, in preparation), ( I ) promotes the expansion of
very early human progenitor cells over 8 weeks of culture
without the addition of human growth factors and (2) stimulates, in synergy with human growth factors, the development of primitive progenitors in short-term semisolid assays,
with some of these progenitors showing a high secondary
recloning capacity.
The first evidence for the selective action of MS-5 on early
human progenitor cells was provided by analysis of the colony-forming ability of the different CD34/CD38 sorted cell
populations in response to growth factors when MS-5 cells
were included in methycellulose short-term assays. In our
experience, as in that of others," the CD38 was the most
powerful antigen to directly separate in one step the clonogenic cells (CD34+/CD38+)from the more primitive pluripotent cells (CD34++/CD38-) contained within the CD34
population. CD34++/CD38- cells, which are free of mature
progenitors and included nearly exclusively primitive progenitors, required at least the three-factor combination of SF
IL-3 Epo to form colonies. This requirement for multiple
cytokines is characteristic of primitive cells and has been previously reported for CD34'Lin- or CD34+/HLADr- human
cells or Scal +Lin- murine ~ells.~'-~'
MS-5 cells strikingly synergized with either A-LCM Epo or IL-3 SF + Epo to
increase fourfold the cloning efficiency of CD34++/CD38progenitor cells as well as colony size. In contrast, MS-5 cells
alone did not stimulate colony formation. Increased cloning
efficiency of CD34++/CD38- was almost entirely accounted
for by increased numbers of BFU-E and CFU-GEMM and
by the growth at days 20 through 22 of small colonies composed of undifferentiated cells. The progenitor cells generating
those colonies that could be classified as blast cell colonies
represented 1% to 2% ofthe CD34++/CD38- population and
generated high numbers of erythroid and granulopoietic secondary colonies upon retransplantation, although there was
no evidence for self-renewal. Very few blast colonies were
detected in the absence of MS-5, even when SF, IL-3, and
Epo were added to the cultures and their replating potential
was very low (Coulombel et al, unpublished observations).
In contrast, MS-5 did not increase the cloning efficiency of
CD34+/CD38+mature clonogenic cells (data not shown).
When CD34++/CD38- cells were grown in long-term cultures using MS-5 as a feeder layer, no human growth factor
was required to maintain hematopoiesis. In each coculture,
the absolute number of clonogenic progenitor cells recovered
after 5 weeks was 2- to 10-fold higher than the progenitor
input numbers, an expansion that is rarely encountered in
classical long-term cultures established on human feeders (this
study, Coulombel et a1,6 and Sutherland et a17).These data
suggest that murine stromal cells might be more effective
than human marrow stromal cells in maintaining progenitors
+
+
+
+
with a high proliferative potential for extensive periods of
time (up to 3 months in our study). Assessment of the number
of clonogenic cells detected at weeks 5 through 8 in cocultures
initiated with 2 to 100 CD34++/CD38- cells showed that
amplification of the immature compartment by MS-5 cells
resulted from the proliferation of a subpopulation representing at least 5% of the CD34++/CD38- cell, with each of
these generating on average 3 to 4 clonogenic progenitor cells.
This frequency is very close to that recently reported for
CD34++/HLADr'" I ' and CD34+/Thyl+ cells.L9We (this
study) observed, as have others," a wide variation in the
number of clonogenic cells generated by CD34++/CD38- cells
cultured at 2 cell-limiting dilution (from 1 to 29 progenitors
per positive well). This must reflect wide variations in the
proliferative capacity of individual primitive stem cells. It is
noteworthy that most of the clonogenic cells produced in our
long-term cultures exhibited characteristics of primitive progenitors with either high proliferative capacities or multipotent potentialities or both. This was in contrast with the
very low output of mature 3- to 8-cluster BFU-E, day-7 to
- 10 CFU-GM, and terminally differentiated cells, indicating
that MS-5 cells did not support human terminal differentiation. Both granulopoietic and erythropoietic progenitors were
produced in almost the same proportion, whereas human
feeders preferentially amplify granulopoietic progenitor
cell^.^^'^ Therefore, these results show that the MS-5 cell line
sustains long-term human hematopoiesis by promoting the
commitment, proliferation, and differentiation into early
granulopoietic and erythroid progenitors of a small population of primitive stem cells. Further studies are required to
understand whether MS-5 cells also have the ability to induce
the differentiation of CD34++/CD38- cells towards other
myeloid and lymphoid lineages, as recently reported for the
B lineage with another murine stromal cell line."
Little is known on how murine stromal cells stimulate
human cell proliferation, but the involvement of yet unidentified growth factors is very likely. Indeed, among hematopoietic growth factors acting on human primitive progenitor
cells, murine IL-6, leukemia-inhibitory factor (LIF), IL-3,
and GM-CSF had no effect.36Only SF and G-CSF are clearly
cross-reactive.36337
MS-5 cells do produce SF," but recent
data suggest that c-kit and its ligand do not play a major role
in the long-term proliferation of primitive murine stem cells
on a stromal cell line.38However, it cannot be totally excluded
that MS-5 acts through membrane-bound growth factors,
which may be more active than their soluble counterpart^.^^
Sustained expansion of primitive progenitors on MS-5 could
also be due to the combined action of growth factors with a
peculiar component(s) of the extracellular matrix, as recently
shown for thrombospondin.40However, our observation that
MS-5 clearly synergizes the action of most recombinant human growth factors, including SF, not only in direct close
contact, but also in short-term assay (methylcellulose culture)
strongly argues for a new presumably diffusible cross-reactive
activity.
The chimeric suspension culture described in this study
allows for extensive periods of time the expansion of adult
human marrow primitive pluripotent hematopoietic progenitor cells without stimulating their terminal differentiation.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
MURINE STROMAL CELLS SUPPORT HUMAN STEM CELLS
As these cocultures are established in the absence of h u m a n
growth factors they will be of particular interest in identifying
new molecules triggering early pluripotent stem cell proliferation and in unraveling the complex interactions between
cytokines and extracellular matrix components that might
alter differentiation and self-maintenance of early human
stem cells. In addition, the synergistic action of MS-5 cells
in clonogenic assays may offer a practical and rapid assay for
evaluating primitive hematopoietic stem cells.
ACKNOWLEDGMENT
We are indebted to P. Lansdorp for his kind gift of the CD34
(8612) antibody, to K. Mori for providing the MS-5 cell line, and
to Amgen for their gift of the rhuSF. We also thank Drs Brunet,
Lapresle, Missenard (Clinique Arago, Paris, France), Koechlin, and
Bonnet (HBpital de la Croix-Rouge, France) for providing marrow
samples. We are grateful to F. Sainteny, A. Dubart, and D. Dumenil
for helpful discussion.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1993 81: 2916-2924
A murine stromal cell line allows the proliferation of very primitive
human CD34++/CD38- progenitor cells in long-term cultures and
semisolid assays
C Issaad, L Croisille, A Katz, W Vainchenker and L Coulombel
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