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Bone Marrow Stromal Cells Induce Myeloid and Lymphoid Development
the Sorted Hematopoietic Stem Cells In Vitro
of
By Ryuhei Okuyama, Masahito Koguma, Nobuaki Yanai, and Masuo Obinata
Regulation of development
of hematopoietic stem cells
was
examined by culturing Lin- c-Kit+ Scal' stem cells sorted
from bonemarrow (BM) cells by fluorescence-activated cell
sorting on a layer ofTBR59, a BM stromal cell line established from simian virus40 T-antigen gene transgenic mice.
The sorted stem cells did not show self-renewal, but two
waves (at 7 and 13 days) of a cobblestone formation were
induced by the stromal cell layer. The cobblestones were
formed by finite cell division(eight divisions on average) of
sorted
Linc-Kit+
Scal' stem cells, anddivided
cells
were still immature. The c-Kithighstem cell population was
induced to form the first wave of cobblestone formation
committed to myeloid lineage, whereas c-Kit'O" population
was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations
require c-Kit function,
but very late activation antigen-&vascularcelladhesion
molecule-l interactionplaysdifferent
parts in the two lineages.
Q 1995 by The American Society of Hematology.
H
attempts, the lineage markers of the differentiated hematopoietic cells were used for negative selection, and the surface
markers of the immature stem cells recognized by specific
monoclonal antibodies (MoAbs) were used for positive selection.22,2'In this work, we examined whether the established stromal cells can support proliferation and differentiation of the purified hematopoietic stem cells by cell sorting.
We showed that particular BM stromal cells can support
induction of development of bothmyeloidand lymphoid
lineages of the sorted stem cells without self-renewal.
EMATOPOIETIC microenvironment is created by the
stromal cells in bone marrow (BM) as shown by a
long-term BM culture, in which the adherent stromal cells
support continued proliferation and differentiation of hematopoietic cells of multilineages in addition to the maintenance of multipotential stem
In this culture, a variety
of stromal cells including endothelial cells, macrophages,
fibroblast cells, and preadipocytes may support stem cells
and their progenitor cell^.^,^ The stromal cells may function
by direct cell-to-cell contact with hematopoietic cells,'"
expressing extracellular matrices,'"' and factors controlling
Proliferationanddifferentiationofthehematopoietic
cell^.'"'^
However, because of the mixed population of the BM stromal cells, it is difficult to examine the cellular and molecular
events involved in the regulation of hematopoiesis. Several
attempts were made to show that theestablished stromal cell
lines from the BM could support long-term maintenance of
stem cells and progenitors.'.''.''
To know how the stromal cells create a hematopoietic
microenvironment and regulate proliferation and differentiation of the stem cells at the cellular and molecular levels,
a variety of stromal cell lines mustbe established and a
microenvironment reconstructed in vitro that is adequate for
hematopoietic regulation. For this purpose, we established
many stromal cell lines from BM using temperature-sensitive
(ts) T-antigen transgenic mice, by which we could immortalize many cell lines with differentiated functions present in
the original tissue.'"*' In addition, the stem cells have to be
purified to examine the direct cell-to-cell interaction between
them and stromal cells. Several attempts were made to purify
the hematopoietic stem cells using a cell sorter; inmost
From the Department of Cell Biology, Institute of Development,
Aging and Cancer, Tohoku University, Seiryu-machi 4-1. Aoba-ku,
Sendai 980, Japan.
Submitted February 6, 1995; accepted June 6, 1995.
Supported byaGrant-in-Aidfrom
the Ministry of Education,
Science, and Culture of Japan.
Address reprint requests to Masuo Obinata, PhD, Institute of
Development, Aging and Cancer, Tohoku University, Seiryo-machi
4-1, Aoba-ku, Sendai 980, Japan.
The publication costsof this article were defrayedin 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 1995 by The American Society of Hematology.
0006-4971/95/8607-0032$3.00/0
2590
MATERIALS AND METHODS
cytukines. Anti-B220 (RA3-6B2, kindly provided by Dr H. Yagita, Juntendo University School of Medicine,
Japan), anti-c-Kit antibody (ACK2, kindly provided byDr S.-I.
Nishikawa, Kyoto University, Kyoto, Japan),u Sca-l (an antibody
specific for the Ly-6AfE molecule; clone E13 161-7, kindly provided
by Dr Y. Aihara, Yokohama City University, Yokohama, Japan),"
TERl19 (erythroid lineage marker; kindly provided by Dr T. Kina,
Kyoto University, Kyoto, Japan), Mac1 (M1/70; Caltag Laboratories, South San Francisco, CA), and three lineage markers
[Grl(RB6-8C5), L3T4 (GK1.5), Ly-2 (53-6.72); Pharmingen, San
Diego, CA] were purchased or provided as indi~ated.~'
Antibody
PS/2, which recognizes mouse a 4 subunit of very late activation
antigen-4 (VLA-4), and antibody WK2, which recognizes mouse
vascular cellular adhesion molecule-l (VCAM-l) were gifts of Dr
H. Yagita.26,27
Murine recombinant stem cell factor (SCF), murine
recombinant interleukin-3 (IL-3). and human recombinant IL-6 were
generously supplied by KIRIN-Amgen CO, Ltd (Tokyo, Japan).
Maintenance of BM stromal cells. Mouse BM stromal cell lines
(TBR series) were established from ts simian virus 40 (SV40) Tantigen transgenic mice as described previous1y.l' TBR cell lines
were maintained in RITC 80-7 (Kyokuto Pharmaceutical CO, Ltd,
Tokyo, Japan) mediumZSas described previously.' The cultures were
incubated at 3YC, which is a permissive temperature for SV40 ts
T-antigen.
Enrichment of hematopoietic Stem cells. A highly enriched stem
cell fraction was obtained from BMs of C57BW6 mice by the method
of Okada et alZ3with a slight modification. BM cells were incubated
with a cocktail of MoAbs, specific to lineage markers (B220, Macl,
Grl, L3T4, Ly-2, and TERl19). Lin+ cells were eliminated by immunomagnetic beads (Dynabeads M-450, coated with antirat IgG,
Dynal AS, Oslo, Norway). Quality of Lin- cells was monitored
by fluorescence-activated cell sorting (FACS) analysis with several
lineage markers. Contamination of either Grl+ or Macl' cells was
less than 1 % and B220', Macl+, TERl19', CD4+, CD8' cells were
not detected. Lin- cells were successively incubated with fluorescein
isothiocyanate-conjugated Scal and biotinated ACK2, phycoerythrin-streptavidine (Pharmingen), and propidium iodide. c-Kit'
Antibodiesand
Blood, Vol 86, No 7 (October l ) , 1995: pp 2590-2597
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MYELOID AND LYMPHOIDDEVELOPMENT OF STEMCELLS
Scal+cells were analyzed and sorted on a FACStar (Becton Dickinson, San Jose, CA) with an automated cell deposition unit.
Coculture ofthe sorted stem cells onthestromal
cells. For
cocultivation of hematopoietic stem cells or BM cells on stromal
cell lines, confluent cell layers of TBR cell lines were maintained
at 37°C by replacing the maintenance mediumwith a-minimum
essential medium (a-MEM: Flow Laboratories, Irvine, CA) supplemented with 10% fetal bovine serum (FBS) and 50 mmol/L 2mercaptoethanol (2-ME). The medium was changed at 3-day intervals. After seeding of Lin- c-Kit' Scal' cells, dishes were scanned
with an inverted-phase microscope for foci containing more than 10
hematopoietic cells attached to or beneath the stromal cells (cobblestone area).'
In vitro methvlcellulose assay. Methylcellulose assay of the hematopoietic cells was performed according to the method of Iscove
et al" with a slight modification. Hematopoietic cells adhered to the
stromal cells were recovered by dispase ( I X 10' UlmL) and the
recovered cells were resuspended in a-MEM containing 30% FBS,
I % deionized bovine serum albumin, 1.2% methylcellulose,
moln 2-ME, SCF ( 1 0 0 ng/mL), IL-3 (200 U/mL), and IL-6 (20 ng/
mL). After 14 days of culture at 37°C. colonies consisting of more
than 50 cells were counted in triplicate as colony-forming units in
culture (CFU-C), and colonies greater than 2 mm in diameter were
regarded as high proliferative potential colony-forming cells (HPPCFC).'"
Analysis ofcobblestoneformation. The number of cells attached
to or beneath the stromal cells in the cobblestone area was counted
by daily photographing and the maximal colony size was determined.
For histochemical analysis ofthe cells released into the medium
from the cobblestones, cytospin preparations were made and stained
with May-Grunwald-Giemsa. At least 1 0 0 cells were observed for
determination of the content of myeloid and lymphoid cells. For
immunostaining, cytopsin preparations were fixed with acetone and
stained with anti-B220 or ant-Grl antibodies.
Effect of MoAbs on the cobblestone formation. To examine the
effect of c-Kit, VLA4. and VCAM-I on the cobblestone formation,
these antibodies were added at 3-day intervals to the coculture of
the Lin- c-Kit' Scal' cellson the stromal cell layers, and the
cobblestones were counted.
RESULTS
Selection of BM stromal cells to support hematopoietic
stem cells. The establishment and characterization of these
stromal cell lines from temperature sensitive (ts) SV40 Tantigen transgenic mice have been reported previously." To
select BM stromal cell lines that support self-renewal or
proliferation and differentiation of stem cells, 34 BM stromal
cell lines were examined by coculturing BM nonadherent
cells on each stromal cell layer. The long-term maintenance
of cobblestone formation without addition of any cytokine
is shown in Fig 1. The BM stromal cells exhibited different
ability to support long-term maintenance of cobblestones in
culture. The cell line TBR59 showed the longest supporting
ability, and thus, we used this stromal cell line for further
studies.
Generation of CFCs from sorted stem cells on the BM
stromal cells. To examine the effect of the BM stromal
cells on hematopoietic stem cells, BM stem cells were enriched by the cell sorting method of Okada et aI2' because
their Lin- Scal' c-Kit' cells sorted from BM cells were
shown to have capacity to reconstitute hematopoiesis. The
Lin- cells prepared by the magnetic bead adsorption method
2591
Cell Lines
Days
0
10
I
TBR 33
TBR 322
TBR 9
TBR 53
TBR 343
TBR 351
TBR 11
TBR e
PA6
TBR 10-1
TBR B
TBR 91
TBR 59
20
30
40 70
60
50
I
I
I
I
I
U
Days
Cell Lines
0
10
I
20
I
30
40
50
60
I
I
I
I
TBR 184
TBR 152
TBR 52
TBR 32
TBR 512
TBR 54
TBR c
TBR 337
TBR 26-1
PA6
Fig 1. Maintenance of cobblestone formation in cocukure of BM
cells with BM stromal cell (TBR) lines established from ts T-antigen
transgenic mice. Nonadhesive BM cells (1 x 10' cells/flask) were
cocukured with the stromal cell layers in 25-cm2flasks,and presence
of t h e cobblestones was observed by phase-contrast microscopy.
Values are t h e average duration time in two flasks (days) when the
last 1 cobblestone disappeared from t h e cocukure. As a control stromalcellline, the preadipocyte cellline, PA6,15 was included. The
results of 23 among 34 clones examined are shown in t h e figure.
were stained with antibodies against c-Kitand Scal. and
Lin- Scal+ c-Kit+cellswere fractionated by FACS (Fig 2).
Quality of the Lin- cells was confirmed by the absence of
lineage markers in the FACS analysis as described in Materials and Methods and the sorted cells were of medium size
and histologically immature hematopoietic cell type. In addition, no B220' cells were observed among more than 300
sorted stem cells analyzed by immunostaining (data not
shown). The colony-forming ability of the sorted stem cells
in the semi-solid culture showed 35 to 45 colonies (CFU-CS)/
100 sorted cells, which was in agreement with the previous
report.*?In HPP-CFC assay, three to five colonies were generated from 1 0 0 sorted cells. We used these preparations as
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OKUYAMA ET AL
2592
..
jy::.........'.".:
. . : :.. ...
.
.
. . .. .. . .. .. .. .. . .. . .
%::+02:
..: . . . . .
:..:..
i
. .
n' c-Klthigh Seal'
Lln' c-Kltlow Seal+
. .. .. .. .. .. .. .. . .. .. . .
. .. .. .. .. .. .
..
1 ' ' 1 " 1 1 1 1 ' ' 1""1
'
Scal
Fig 2. Purificationof hematopoieticstem cells. Lin- BM cells were
stained with c-Kit and Scal and analyzed by FACStar. Double-positive (Lin' c-Kt' Scal') cells (fractions 4 and 51, Lin- c-Kithigh Scal'
(fraction 5). and Lin-c-Kit'"" Scal+ (fraction 4) were sorted for coculture with thestromal cells.
cell division cycles were 1 1 at most, with an average of 8.
Thus, a cobblestone containing a maximum of 1,000 cells
was generated by the rapid expansion of the sorted cells.
Two waves of cobblestone formation on the stromal cells.
Generation of the cobblestones seemed to have two waves:
one major peakon day 7 and a second peak on day13
(Fig 3A). Cells were released from the cobblestones into the
overlaying medium during coculture on TBR59 stromal
cells. In the medium, the percentage of mature granulocytes
was 80% on day 9 (2 days after the first peak) and slightly
decreased to 70% on day 15 (2 days after the second peak),
whereas the percentage of lymphocytes slightly increased
from day 9 (20%) to day 15 (30%) (Fig 5, A and B). After
30 days of culture, the number of the cobblestones decreased
to = IO, but they were maintained for over 30 days. The two
waves of cobblestone formation implied either the presence
of two cell populations or two-step hematopoietic cell development responding to the stromal cells. We sorted the Lin-cKit' Scal' stem cells into Lin-c-KithiFhScal' (0.16% of
Lin- cells) and Lin-c-Kit'"" Scal '(0.06% of Lin- cells) populations and examined their colony formation on TBR59
stromal cell layers. The result indicated that the c-Kithieh
population formed the first wave of cobblestones and the cKit'"" population formed the second wave (Fig 3B), confirming that the two stem cell populations respond to stimulation
by the stromal cells. The Lin-c-KithishScal' population released mostly granulocytes, whereas the Lin-c-Kit'"" Scal
population released mostly lymphocytes (Fig 5, A and B).
Immunostaining showed that the released cells from c-Kithigh
were Gr1'-granulocytes (Fig X ) , whereas those from the
c-Kit'"" population were B220'-lymphocytes (Fig 5D). The
Lin-c-KithIghScal ' population produced megakaryocytes,
macrophages, and monocytes, most of which were attached
to the stromal cells; few of them were released into the
overlaying medium (data not shown).
Molecules involved in the interaction between the stromal
cells and stemcells that are required for cobblestoneformation. To know molecules involved in the interaction between the stromal cells and stem cells, MoAbs were added
to the coculture. Addition of the anti-c-Kit antibody almost
completely inhibited the two waves of cobblestone formation. The inhibition was observed in both the number (Fig
+
sorted hematopoietic stem cells for the coculture with
TBR59 stromal cells. When the HPP-CFC was measured
during coculture, the value dropped gradually with the coculture (Table 1). Thus, it seemed thatself-renewal of the sorted
stem cells did not occur. However, we were able to identify
the small colonies (CFU-CS) containing more than 50 cells.
The CFU-C increased for 1 week,then fell, and without
stromal cells there was no colony formation. Thus, the stromal cells induced a transient increase in the CFU-C-forming
cells from the sorted stem cells within 7 days of coculture.
We then examined directly the fate of the sorted stem cells
that were on the TBR59 stromal cells.
Cobblestone formation of the sorted stem cells on the
stromal cells. During the coculture, we observed cobblestone formation of the hematopoietic cells. The formation
was observed in three stromal cells among 34 stromal cell
lines (data not shown). The round cells in the hematopoietic
cobblestones were phase dense, indicating they lay underneath or attached to TBR59 cells.' The hematopoietic cells
within the cobblestone area had indented nuclei and no distinct features of mature hematopoietic cells, but were morphologically different from the initial sorted cells. The cobblestone formation was dependent on mostly the stem cell
population because onlyc-Kit'
Scal' cell populations
formed the cobblestones; c-Kit' Scal- and c-Kit- Scal' cell
populations of the Lin- hematopoietic cells rarely formed
cobblestones containing a large number of cells (Fig 3A).
The cobblestone formation continued for more than 30 days
of the coculture. The maximum cobblestone size was measured by counting the number of phase-dense cells in each
cobblestone as shown in Fig 4. In most cobblestones, the
Table 1. Kinetics of Colony-FormingActivities in Cocukure of the
Sorted Stem Cells With TBR59 Stromal Cell Layers
Colonies per 100 Cells (dl
Colony
Type
0
7
14
21
HPP-CFC
CFU-C
5.3 2 0.6
37.3 ? 1.7
3t2
185.3 2 3.4
0.3 2 0.6
88 t 20.7
0?0
65 2 12.2
Onehundredsorted
Lin-c-Kit'Scal' cells were cocultured with
TBR59 stromal cells (8 x lo5 cells/flask). All cellswere removed from
the flasks by dispase treatment at 0,7, 14, and 21 days and seeded
in methylcellulose culture medium supplemented with SCF (100 ng/
mL). IL-3 (200 U/mL) and IL-6 (20 ng/mL). The sorted cells cultured in
the same medium without the stromal cellsdid not form any colonies.
Values are the average of three dishes. HPP-CFU indicates colonies
greater than 2 mm in diameter andCFU-Cindicatescoloniescontaining more than 50 cells.
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MYELOID AND LYMPHOIDDEVELOPMENT OF STEM CELLS
2593
E
3
Z
)r
-c00
0
0
5
10
15
20
Days
1
1207
'L
2O 2'
Cell Number
Fig 4. Number of phase-dense cells in a cobblestone. Pictures
taken daily allowed determination of the maximum number
phase-of
dense cells in each cobblestone; distribution of the cells in a single
cobblestone was shown in 21 cobblestones.
3C) and size (data not shown) of cobblestones. Then the
involvement of the adhesion molecules on the cobblestone
formation was examined. The inhibitory activities of antiVLA-4 antibody were saturated at the concentration of 1 pgI
mL, but anti-VLA-4 antibody inhibited partially (40%) in
the first wave of cobblestone formation even at a high dose
and inhibited completely (80% to 90%)in the second wave
(Fig 6A). The inhibitory effect of this antibody suggested
the presence of ligand molecules for this integrinin the
0
5
10
15
20
Days
80
1
T
60
40
20
00
5
10
Days
15
Fig 3. Cobblestone formation of the sorted
stern cell fractions. (A)
Kinetics of cobblestone formationof the sorted stem cellfractions.
Five hundred cells in each fraction of Lin- population were seeded
on TBR59 cell layer. Cocultures were maintained at
37°C with a-MEM
supplemented with 10% FBSand 2-ME. Foci containing more than10
phase-dense cells were counted as cobblestones by inverted-phase
microscopy. Lin- c-Kit+ Scal' fraction(0)
generated cobblestones in
two waves (days 7 and 13). Lin c-Kit' Scal IO I and Lin-c-KitScal' (0)
fractions showed low cobblestone formation and Lin cKit- Scal- IAl formed nocobblestones. The values were taken from
three independent experiments. IBI Cobblestone-forming activities
of c-Kithighand c-Kt'"" populations of Lin- Scal+ cells. Five hundred
cells of each of Lin- c-Kit+ Scal'. Lin- c-Kithigh Scat' and LincKit'""' Scal' populations were cocultured on
TBR59 cell layer and
cobblestones were counted. Lin- c-Kit+Scal+ IO)showed two peaks
of cobblestone formation, whereas Lin- c-Kthigh Scal' ( 0 I showed
showed a single
a single peak on day 7 and Lin- c-Kit'''" Scal' (0)
peak on day 11. Values are the average of four dishes from two
independent experiments. IC) The effect of anti-c-Kit antibody on
cobblestone formation. Five hundred Lin- c-Kt' Scal+
cells were cocultured on TBR59 cell layers. c-Kit antibody was added at 0 IO), l
( 0), 10 (0)
or 50 (A)pg/mL. Ateach medium change 13-day intervals),
the antibody was added t o the culture and the cobblestones were
counted. Values are the mean oftwo dishes.
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OKUYAMA ET AL
2594
A
Percentage
9 Days
0
20
40
60
80
100
Granulocyte
stromal cells. Expression of VCAM-I, a ligand for VLA-4
was confirmed by FACS analysis (data not shown). Addition
of anti-VCAM- 1 caused was essentially the same reduction
as did anti-VLA-4; its inhibition was weak in the first wave
(50% to 60%) but strong in the second (80% to 90%) at the
concentration of 1 pg/mL (Fig 6B). However, an increased
dose of anti-VCAM-l completely inhibited even thefirst
wave. The cobblestones inhibited by VLA-4 or VCAM-I
antibody contained a small number of phase-dense hematopoietic cells with notable morphologic difference from that
of c-Kit antibody (data not shown).
Monocyte
DISCUSSION
l Macrophage
L
Lymphocyte
B
Percentage
15 Days
0
Granulocyte
Monocyte
l Macrophage
r
b
20
40
60
80
100
By definition, stem cells have extensive capacity for selfrenewal that extends throughout the life of the organism
and, thus, is essentially unlimited. In contrast, the number
of rounds of divisions that a transient amplifying cell undergoes isfinite; transient amplifying keratinocytes undergo
terminal differentiation within 15 cell generations in culture": the 0-2A progenitor cells of rat optic nerve undergo a
finite number of divisions in the presence of platelet-derived
growth factor-produced by type 1 astrocytes before differentiating into oligodendrocytes.'* The mechanism that determines the number of divisions in these situations is unknown,
but one possibility is that the cells are able to count cell
divisions and after an appropriate number undergo terminal
differentiation.32.33
In the present work, weshowed that an established stromal
cell line, TBR59, can induce a finite number of divisions
committed to myelopoiesis and lymphopoiesis of the BM
hematopoietic stem cells sorted as Lin- c-Kit' Scal' cells.*'
This finite division was observed by a cobblestone formation
in the absence of exogenously added soluble factors and was
promoted by particular stromal cells established from BM
because we found that only three stromal cells among 34
established stromal cell lines that could induce cobblestone
formation of the sorted stem cells. We established many BM
stromal cell lines from ts T-antigen transgenic mouse that
showed characteristics of endothelial cells, preadipocytes,
and fibroblasts," but in our analysis, the stem cell supporting
ability was only observed in three preadipocyte cell lines
(data not shown). In TBR59 cells, adipocyte conversion was
induced at 39°C or in the presence of dexamethasone (data
not shown). Even at 37"C, TBR59 cells differentiated
slightly to adipocytes, and a slight temperature shift from
37°C affected the stem cell supporting ability (R. Okuyama,
Fig 5. Characterization of the differentiated blood cells from cand c-Kd- populations of Lin- Scal' cells. Sorted cells were
cocultured on the TBR59 cell layers.
The hematopoieticcells released
to the overlaying medium from the coculture of Lin- c-Kit'
Scal'
(0).Lin- c-Kithigh
Scal (01,and Lin- c-Kit" Scal' IO)were recovered
by centrifugation onto glass slides at day 9 (A) and day 15 (B) and
stained with May-Grunwald-Giemsa.Characterizationof the differentiated blood cell types was performed by microscopic observation of
more than 100 cellsin every fraction. In addition, released cellswere
immunologicallystained with anti-Grlantibody (C) or B220 antibody
ID).
Kith'sh
+
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2595
MYELOID AND LYMPHOIDDEVELOPMENT OF STEMCELLS
A
"
I
0
T
5
10
15
20
15
20
Days
a,
v)
0
.c
00
0
m
0
5
10
Days
Fig 6. The affects of anti-VLA-4 and VCAM-1 antibodies on cobblestone formation. Lin- c-Kit+ Scal+ cells were cultured on TBR59
cell layers. The cobblestones were formed in the presence of either
antibody PS/2 (anti-VLA-4) (A) or MIK2 (anti-VCAM-l) (B). Antibodies were added at the concentration of 0 (0).
1 I O 1, 10 ('3)or 50 (A)
pg/mL. Values are the average of two dishes.
unpublished observation, June 1994), suggesting that the differentiated phenotype of the stromal cells is important in
regulation of the stem cells.
We observed two waves of cobblestone formation from
Lin- c-Kit' Sca-l' stem cells; the first wave was derived
from the c-Kithigh
population, whereas the second was from
the c-Kit'"" population. The mature cells released from cKithighpopulation were mostlymyeloid lineages. whereas
those from c-Kit'"" population were mostly lymphocytes,
indicating that the c-Kit'"ghstem cell population may be com-
mitted to myeloid lineages on the stromal cells, whereas
the c-Kit'"" population may be committed to lymphopoiesis.
Because B220' lymphoid progenitor cells were not detected
in the sorted stem cells, lymphopoiesis maynotbe caused
by expansion of the contaminated lymphocyte progenitors
in the absence of hydrocortisone. In addition, supplementation of IL-3 to the coculture-stimulated cobblestone formation in the first wave with an increase in the released cells
of myeloid lineage, but was not stimulatory for the second
wave (R. Okuyama, unpublished observation, August 1994).
Because a previous report suggested that the c-Kit'"" population contains more immature stem cells," our result could
not exclude the possibility that c-Kit'"" population starts developing at a later time point andhas the potential to generate
both lymphoid and myeloid cells, but it is more likely that
the two populations have different potentials for the lineage
commitment. This is the first demonstration of clear separation of the stem cell populations into lymphopoiesis and
myelopoiesis. We recently found that another stromal cell
line also predominantly supported lymphopoiesis of the
sorted stem cells (Koguma et al. manuscript in preparation).
These coculture systems will be useful for analysis of commitment of the hematopoietic stem cells in vitro.
Whereas myelopoietic and lymphopoietic development of
the stem cells could beinduced by the stromal cells, we
were unable to induce self-renewal of the stem cells by the
stromal cells, because their HPP-CFC formation dropped
rapidly during coculture. This is consistent with previous
reports that the sorted stem cells cannot self-renew, even in
the presence of stromal cells,'5.'6 although Fraser et aI3' reported that the stem cells can self-renew in vitro on the BM
stromal cells. There may be several possible reasons for the
inability of sorted stem cells to self-renew: ( l ) the stromal
cells that can support self-renewal of the sorted stem cells
have not yet been obtained, and thus, more BM stromal cells
must be screened of this ability; (2) self-renewal may require
a mixture of stromal cells with different abilities (although
this does not seem likely from the previous report on Dexter's culture?" the possibility is testable by a mixed culture
of a variety of established stromal cells); (3) in addition to
the stromal cells, a factor produced by other blood cells may
be required for stem cell self-renewal.
We examined molecules involved in the interaction between the stromal and stem cells. c-Kitis thought to be
essential for stem cell pr~liferation""~"
and we showed that
c-Kit is essential for cobblestone formation; thus, c-Kit and
its ligand, SCF, whose expression in TBR59 cells was confirmed by reverse-transcriptase polymerase chain reaction
(R. Okuyama, unpublished observation, January 1995), are
essential for proliferation of the sorted stem cells on the
stromal cells. The importance of adhesive interactions in
hematopoiesis has beenwell studied in lymphoid organs;
integrins VLA-4,VLA-5.and VLA-6 are responsible for
proliferation and activation of lyrnpho~ytes.4'~~'
The involvement of VLA-4"' andV-CAM-I"." in the association of
hematopoietic stem cells and stromal cells as well as in the
stroma-dependent erythropoiesis' has recently been reported.
We confirmed the expression of VCAM-I in TBR59 cells
by FACS analysis (data not shown). Antibody against VLA-
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2596
OKUYAMA ET AL
4 inhibited cobblestone formation in the first andsecond
waves, but interestingly, in the firstwave (myelopoiesis),
inhibition of VLA-4 was partial, whereas that of VLA-4 was
complete in the second wave (lymphopoiesis). The inhibition
of antibody against VCAM-1, a ligand for VLA-4, gave
essentially the same result, suggesting that VLA-4-VCAM1 interaction participates more strongly in lymphopoiesis
thanin myelopoiesis. It islikely that in myelopoiesis the
hematopoietic cells must adhere to the stromal cells through
VLA-4-VCAM-I interaction, but their continuous interaction is not required for further proliferation and differentiation of the hematopoietic cells. However, their continuous
interaction may be required for lymphopoiesis. Because expression of SCF and VCAM-I may not be restricted to the
stromal cells that induced the cobblestone formation, they
alone may not be sufficient to support the sorted stem cells,
although their expression is essential. The search for the
molecules whose expression is restricted to TBR59 cells may
be important for understanding the molecules involved in
the stromal-stem cell interaction.
In conclusion, we established a new in vitro system for
myelopoietic and lymphopoietic development of sorted stem
cells using cloned BM stromal cells. This system willbe
valuable for analyzing the mechanism of lineage commitment of multipotent hematopoietic stem cells in vitro at the
cellular and molecular levels.
ACKNOWLEDGMENT
We thank Dr K. Kumagai for support of FACS analysis, and Drs
S.-I. Nishikawa, Y. Aihara, and H. Yagita for MoAbs.
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1995 86: 2590-2597
Bone marrow stromal cells induce myeloid and lymphoid
development of the sorted hematopoietic stem cells in vitro
R Okuyama, M Koguma, N Yanai and M Obinata
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