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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 923-931
The Soluble Interleukin-6 (IL-6) Receptor/IL-6 Fusion Protein Enhances
In Vitro Maintenance and Proliferation of Human
CD34+CD38 /low Cells Capable of
Repopulating Severe Combined Immunodeficiency Mice
By
Orit Kollet,
Ronit Aviram,
Judith Chebath,
Herzl ben-Hur,
Arnon Nagler,
Leonard Shultz,
Michel Revel, and
Tsvee Lapidot
From the Departments of Immunology and of Molecular Genetics, The
Weizmann Institute of Science, Rehovot, Israel; the Department of
Obstetrics and Gynecology, Kaplan Hospital, Rehovot, Israel; the
Department of Bone Marrow Transplantation, Hadassah University
Hospital, Jerusalem, Israel; and The Jackson Laboratory, Bar
Harbor, ME.
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ABSTRACT |
In vitro maintenance and proliferation of human hematopoietic stem
cells is crucial for many clinical applications. Early hematopoietic
cells express low levels of FLT-3 and c-kit receptors, as well as the
interleukin-6 (IL-6) receptor signal transducing element, gp130, but do
not express IL-6 receptor itself. Therefore, we have attempted to
maintain human cord blood or bone marrow CD34+ cells ex
vivo in serum-free cultures containing stem cell factor (SCF) and FLT-3
ligand (FL) alone or together with a new recombinant molecule of
soluble IL-6 receptor fused to IL-6 (IL6RIL6 chimera). The effect of
IL6RIL6 chimera on the proliferation and differentiation of
CD34+ cells was compared with that of each chimera
component added separately. The engraftment potential of in
vitro-cultured cells was determined using our recently established
functional in vivo assay for primitive human severe combined
immunodeficiency (SCID)-repopulating cells (SRC). We
report here that IL6RIL6 chimera induced significantly higher levels of
progenitors and SRC compared with SCF + FL alone or together with
IL-6 and soluble IL-6 receptor. IL6RIL6 chimera prolonged in vitro
maintenance of SRC for up to 14 days. Stimulation of
CD34+CD38 /low enriched cells with IL6RIL6
chimera maintained the early CD34+CD38/low
cell subpopulation, which could be detected in vitro for up to 14 days.
Moreover, IL6RIL6 chimera preferentially stimulated the growth of early
CD34+38/low cells, resulting in
significantly higher levels of progenitors compared with more
mature CD34+38+ cells. Taken together,
these findings demonstrate the importance of IL6RIL6 chimera in
stimulating the proliferation of early
CD34+· CD38gp130+IL-6R
cells in vitro and extended maintenance of progenitors and SRC.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC stem cells continuously
produce all mature blood cells by extensive proliferation and
multilineage differentiation. Human hematopoietic CD34+ or
CD34+CD38 cells, which are enriched for
stem cells, play a major role in the long-term bone marrow (BM)
reconstitution of ablated patients in autologous and allogeneic
transplantations. BM transplantation and other clinical applications,
such as purging of tumor cells and gene therapy, involve in vitro
maintenance of CD34+ cells. Thus, many studies are focused
on defining appropriate conditions for culturing CD34+
cells in vitro, with particular emphasis on their engraftment and
repopulation potential. Several groups have used human and murine
stromal cells to support the maintenance in vitro of CD34+
or CD34+CD38 cells.1-3
Others replaced the stromal cells and their soluble secreted factors
with recombinant human cytokines to produce a microenvironment capable
of maintaining the primitive cells.4-8
The early acting cytokines stem cell factor (SCF), FLT-3 ligand (FL),
and interleukin-6 (IL-6) are commonly used to maintain hematopoietic
stem cells in vitro. SCF and FL were shown to improve survival and
maintenance of hematopoietic progenitors.9,10 The addition
of IL-3 resulted in the expansion of human long-term culture-initiating
cells (LTC-IC) in vitro.5,11 IL-6 is a potent cofactor for
the survival and proliferation of primitive multilineage progenitor
cells in liquid cultures in vitro.12,13 Moreover, defective
hematopoiesis in IL-6-deficient mice indicates that IL-6 plays such a
role in vivo as well.14 IL-6 acts on cells through a
receptor system comprising 2 proteins, IL-6 receptor (IL-6R, gp80) and
gp130.15,16 Signal transduction is solely due to
dimerization of gp13017 upon formation of a hexameric complex of 2 gp130, 2 IL-6R, and 2 IL-6 ligands.18-20
Soluble forms of IL-6R (sIL-6R) are produced by cells and are found in
blood and urine.21,22 These sIL-6R act as a potent agonists
of IL-6 on many cell types,15,23 because they retain the
ability to induce IL-6-dependent gp130 dimerization. Stimulation of
human cord blood (CB) CD34+ cells by adding sIL-6R together
with IL-6 to SCF24 or to FL25 led to marked in
vitro stimulation of multilineage progenitors, followed by their
expansion. CD34+ cells expressing gp130 but not IL-6R
represent a more primitive cell population enriched for LTC-IC that can
be stimulated when both sIL-6R and IL-6 are added to SCF and
IL-3.26,27 By fusion of sIL-6R to IL-6, chimeric IL6RIL6
proteins were obtained that have a higher activity than the mixture of
IL-6 + sIL-6R for in vitro expansion of early hematopoietic progenitors
when added to SCF + IL-328 or to SCF + FL, as we recently
showed.29
In previous studies, primitive human stem cells could not be maintained
in vitro successfully for long periods of time due to differentiation.
In addition, the repopulating potential of cultured primitive cells
cannot be assayed in vitro. These difficulties led to the establishment
of several animal models, thus facilitating the study of human stem
cells development and biology.30-32 We have previously
described a functional in vivo assay for primitive human hematopoietic
cells based on their ability to repopulate the BM of sublethally
irradiated SCID mice homozygous for the severe combined
immunodeficiency Prkdcscid mutation and more
recently nonobese diabetic (NOD)/SCID mice, after
intravenous transplantation. We have defined the engrafting human cell
as an SCID-repopulating cell (SRC).33,34 Purification assays demonstrated that SRC are phenotypically characterized as
CD34+CD38 cells.34 Recent
studies provide evidence that
CD34CD38 cells35 and
CD34+CD38+6 cells also have limited engraftment
potential. Kinetic studies showed that only a small fraction of the
transplanted cells engraft and repopulate the murine BM by extensive
proliferation and multilineage differentiation.36
Previous results with a cytokine cocktail that included IL-6, SCF, and
FL quantified in 2 similar model systems showed a 2-fold increase in
the levels of SRC after 4 days7 or competitive repopulating
units (CRU) after 5 to 8 days6 of in vitro stimulation. However, on day 9, in vitro analysis demonstrated the loss of CD34+CD38 cells, and SRC could not be
detected.7
In the present study, we investigated the potential of IL6RIL6 chimera
to maintain and expand ex vivo SRC capable of repopulating the BM of
NOD/SCID mice.
We describe here stroma-free culture conditions for human SRC
maintenance in ex vivo cultures established for up to 14 days while
maintaining and enhancing their repopulating potential. IL6RIL6 chimera
increased the levels of progenitors and SRC by acting mainly on the
primitive CD38/low subpopulation of
CD34+ cells. We suggest an important role for IL6RIL6
chimera in stimulating the proliferation of early human
CD34+· CD38/lowgp130+IL-6R
cells in vitro, resulting in extended maintenance of
progenitors and SRC.
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MATERIALS AND METHODS |
Human cells preparation.
Human CB samples were obtained from full-term deliveries after informed
consent was obtained. Human BM cells were obtained from harvests of
normal donors for allogeneic transplantation after informed consent.
Human cells were used in accordance with the procedures approved by the
human experimentation and ethics committees of the Weizmann Institute
(Rehovot, Israel). The blood samples were diluted 1:1 in
phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum
(FCS), without Mg2+/Ca2+. Low-density
mononuclear cells (MNC) were collected after standard separation on
Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and washed in RPMI
with 1% FCS. Some samples were frozen in 10% dimethyl sulfoxide
(DMSO), whereas the others were used fresh. Enrichment of
CD34+ cells was performed with mini MACS separation kits
(Miltnyi Biotec, Bergisch Gladbach, Germany) according to the
manufacturer's instructions. The purity of the enriched
CD34+ cells was 60% to 80% using 1 column and greater
than 97% when cells were passed over 2 columns.
CD34+CD38 enrichment was performed
either by a StemSep kit (StemCell Technologies Inc, Vancouver, British
Columbia, Canada) according to the manufacturer's instructions or by
fluorescence-activated cell sorting (FACS; FACStar+; Becton Dickinson, San Jose, CA)
after staining with monoclonal antibody (MoAb) antihuman
CD34-fluorescein isothiocyanate (FITC; Becton Dickinson) and antihuman
CD38-phycoerythrin (PE; Coulter, Miami, FL). The purity of the obtained
subpopulations was 55% to 65% (StemSep) and greater than 99% (FACS), respectively.
Mice.
NOD/SCID mice were bred and maintained under defined flora conditions
at the Weizmann Institute in sterile micro-isolator cages. All of the
experiments were approved by the animal care committee of the Weizmann
Institute. Sublethally irradiated (375 cGy, at 67 cGy/min), 8-week-old
mice were transplanted with human cells as previously
described,33,34 with minor modifications. Briefly, human
cells were injected into the tail vein of irradiated mice in 0.5 mL of
RPMI with 10% FCS. Nonengrafting CD34 carrier cells
were irradiated (1,500 cGy) and were cotransplanted with cultured cells
at a final concentration of 0.5 × 106 cells/mouse.
Mice were killed 1 month after transplantation, and BM cells were
flushed from the 8 bones of each mouse (femures, tibias, humeri, and pelvis).
Ex vivo cultures.
Human CD34+ enriched cells were cultured in 24-well plates
(1 to 2 × 105 in 0.5 mL) containing 10% FCS and 1%
bovine serum albumin (BSA; Sigma, St Louis, MO) or in serum-free media
composed of IMDM, 2% BSA, 20 µg/mL human insulin (Biological
Industries, Beit Haemek, Israel), 40 µg/mL human low-density
lipoprotein (LDL) (Sigma), 200 µg/mL human transferrin
(Sigma), 104 mol/L 2-mercaptoethanol
(2ME), and 10 mmol/L HEPES buffer (pH 7.3).
CD34+CD38/low cells were cultured in
serum-free media in 96-well plates. Ex vivo cultures contained
combinations of the following cytokines: SCF at 100 ng/mL, FL at 100 ng/mL, IL-15 at 100 ng/mL (R&D Systems Inc, Minneapolis, MN),
recombinant human IL-6 (rhIL-6) at 50 ng/mL, and sIL-6R at
1,280 ng/mL (InterPharm Laboratories, The Ares-Serono Group, Ness
Ziona, Israel).29 The fused protein of soluble IL-6 receptor and IL-6 (IL6RIL6 chimera) was produced as
described29 and used at 150 ng/mL. For natural killer
(NK) cell development, BM cells from engrafted mice were
cultured with serum-free media that contained 100 ng/mL of SCF and
IL-15 (R&D) for 10 days. The cultures were incubated at 37°C in a
humidified atmosphere containing 5% CO2.
Colony-forming unit (CFU) assay.
To detect the levels of human progenitors after cytokine stimulation in
ex vivo cultures and in the marrow of transplanted mice, semisolid
cultures were performed as previously described.33,34 In
brief, the cells were plated in 0.9% methylcellulose (Sigma), 30%
FCS, 5 × 105 mol/L 2ME, 50 ng/mL SCF, 5 ng/mL
IL-3, 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF;
R&D), and 2 U/mL erythropoietin (Orto Bio Tech, Don Mills,
Ontario, Canada). Human progenitors from engrafted mice
were plated in 15% FCS + 15% human plasma together with the human
cytokines listed above. These conditions are selective for human
colonies. Plating concentrations were as follows: for enriched
CD34+ cells, 4 × 103 cells/mL; for
CD34+CD38/low cells, 800 cells/mL; and
for BM cells from transplanted mice, 2 × 105
cells/mL. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 and scored 14 days later for
myeloid, erythroid, mixed, and blast colonies by morphologic criteria.
Flow cytometry analyses.
The purity of enriched subpopulations after magnetic beads separation
was analyzed using two-color staining using MoAb antihuman CD34-FITC
(Becton Dickinson) and antihuman CD38-PE (Coulter). The levels of human
cells in the marrow of engrafted mice were detected by staining with
MoAb antihuman CD45-FITC (Immuno Quality Products, Groningen, The
Netherlands). Human pre-B cells were detected by double staining with
MoAb antihuman CD45-FITC/CD19-PE, and NK cells were detected by
antihuman CD45-FITC/CD56-PE (Coulter). Analysis of human gp130 was
performed using anti gp130 (AM64; Pharmingen, San Diego, CA) and MoAb
anti-IL-6 receptor as previously described.37 Human Fc receptors were blocked
using human plasma (1:50) and murine Fc receptors by antimouse
CD16/CD32 MoAb (Pharmingen). Isotype control antibodies were used to
exclude false-positive cells (Coulter). BM cells from irradiated mice
were used as a negative control, and human cells were used as a
positive control. Dead cells were gated out by staining with propidium
iodide (Sigma). Cells were washed with PBS supplemented with 1% FCS
and 0.02% Azide, suspended to a volume of 1 to 5 × 105 cells/mL, stained with direct labeled MoAb, and
incubated for 25 minutes on ice. After staining, cells were washed once
in the same buffer and analyzed on a FACSort (Becton Dickinson).
Analysis was performed using CELLquest software (Becton Dickinson).
Human DNA analysis.
The levels of human cell engraftment were detected as previously
described.33,34 Briefly, high molecular weight DNA was obtained from the BM of transplanted mice by phenol/chloroform extraction. DNA (5 µg) was digested with EcoRI, subjected to
electrophoresis on 0.6% agarose gel, blotted onto a nylon membrane,
and hybridized with a human chromosome 17-specific  -satellite probe
(p17H8) labeled with 32P. After a random digestion with
EcoRI, this probe hybridizes a characteristic multisize band
pattern that is specific for human DNA. For quantification of the human
DNA in the samples, the intensity was compared with that in artificial
mixtures of human and mouse DNA (0%, 0.1%, 1%, and 10% human DNA)
run in parallel lanes. Multiple exposures of the autoradiographs were
taken to ensure sensitivity down to 0.01% human DNA.
Binding of IL6RIL6 chimeric molecule to gp130.
Monoclonal antihuman gp 130 AM64 (2 µg/mL; Pharmingen) was added in a
96-well microplate to 0.1 mL/well of PBS. After 18 hours at 4°C,
the plate was washed and blocked with 1% BSA in PBS (0.1 mL/well) for
3 hours. Soluble gp130 (50 ng/mL; R&D) was added at 0.1 mL/well in PBS
for 2 hours at 20°C. After washing 3 times with 0.05% Tween-20 in
PBS, 0.1 mL of IL-6 (500 ng/mL) with increasing amounts
of sIL-6R (from 4 to 500 ng/mL) were added for 2 hours at
20°C. In other wells, the IL6RIL6 chimeric molecule was similarly added (from 0.1 to 50 ng/mL). After washing, the bound sIL-6R was
quantitated by sandwich enzyme-linked immunosorbent assay (ELISA) with
polyclonal anti-sIL-6R rabbit serum and goat antirabbit Ig-horseradish
peroxidase conjugate.22 Calculations of molar concentrations are based on molecular weights of 60 kD for sIL-6R and
85 kD for IL6RIL6 produced as glycoproteins in CHO cells.29
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RESULTS |
Proliferation and differentiation of human CD34+ cells
cultured ex vivo with SCF, FL, and IL6RIL6 chimera.
In the first experiments, human CB CD34+ cells were
cultured in vitro for 3 or 6 days with SCF and FL to which the IL6RIL6 chimera was added (Fig 1A). On day 3, there
was only a small increase in cell numbers compared with day 0, but on
day 6 there was a 7.5- to 10-fold increase that did not vary
significantly whether SCF and FL were present alone or supplemented
with IL6RIL6 chimera. Similar results were obtained with media
containing 10% FCS or with serum-free media. The proliferation of
cultured CD34+ cells is, therefore, due mainly to SCF and
FL.
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| Fig 1.
Total cell numbers and progenitor levels in ex
vivo-cultured CD34+ cells. CB CD34+
enriched cells were cultured (2 × 105 cells/0.5 mL) in
RPMI + 10% FCS + 1% BSA or in serum-free media, both supplemented
with cytokines, for 3 or 6 days. Cytokines were used in the following
concentrations: SCF at 100 ng/mL, FL at 100 ng/mL, and IL6RIL6 chimera
at 150 ng/mL. (A) Cells were counted for viable cell numbers. (B) Ex
vivo-cultured cells were seeded (4 × 103 cells/mL) into
semisolid media, and colonies were scored on day 14. Progenitor levels
were calculated on the basis of total cell numbers. Values shown are
the mean ± SE from 10 independent experiments.
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Different results were obtained when the expansion of clonogenic
progenitors was determined by plating the ex vivo-cultured CD34+ cells in semisolid media (Fig 1B). Cells that had
been cultured in suspension for 3 days with SCF + FL alone had a
moderate 2-fold increase in the levels of colony-forming progenitors
compared with day 0; however, the increase averaged 8-fold when IL6RIL6 chimera was added. When CD34+ cells were cultured for 6 days, the increase was 5.8-fold with SCF + FL and 21-fold when IL6RIL6
was added. Taken together, IL6RIL6 chimera increased significantly the
levels of progenitors compared with SCF + FL alone after 3 and 6 days
of culture (P = .004 and P = .03, respectively).
Stimulation of SRC by the addition of IL6RIL6 chimera to SCF and FL.
In the next set of experiments, we studied the effect of the IL6RIL6
chimera on maintenance or proliferation of the more primitive SRC in ex
vivo cultures with human CB or BM cells. Enriched CD34+
cells were seeded at 1 to 2 × 105 cells/well and
cultured for 3 or 6 days with various cytokine combinations. At each
time point, the cells of each well were collected and transplanted into
one NOD/SCID mouse by intravenous injection. One month later, the
levels of engraftment were determined by recovering the mouse BM cells
and quantifying the percentage of human DNA by Southern blot analysis.
The highest levels of engraftment were obtained when IL6RIL6 chimera
was added to SCF and FL during the ex vivo cultures of CB and BM cells
(Fig 2A). Figure 2B summarizes the levels
of human cell engraftment in mice (7 per group) that were transplanted
with ex vivo cytokine stimulated cells. As compared with the original
CD34+ cells (day 0), the cells cultured ex vivo for 3 and 6 days showed increases in engraftment that were much higher when SCF and
FL were complemented by the IL6RIL6 chimera. Compared with SCF + FL
alone, the addition of IL6RIL6 chimera increased the engraftment levels
by 9.2-fold at day 3 (P = .0002) and by 6.5-fold at day 6 (P < .0002). Furthermore, in these experiments with total
CD34+ cells, IL6RIL6 chimera was superior to the mixture of
IL-6 and sIL-6R, resulting in higher engraftment levels (Fig 2B).
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| Fig 2.
Quantitative analysis of SRC after 3 or 6 days of ex vivo
cultures. Southern blot analysis of DNA extracted from the BM of
individual NOD/SCID mice transplanted with 105
CD34+ uncultured cells or with their expanded progeny
after ex vivo cultures. (A) Representative Southern blots. BM of mice
transplanted with human BM CD34+ cells cultured for 3 days with indicated cytokine combinations. BM of mice transplanted with
the original CB CD34+ cells before seeding (day 0) or
with cells expanded ex vivo for 6 days with SCF + FL + IL6RIL6
chimera. DNA was extracted from the BM of transplanted mice 1 month
after transplantation and was hybridized with a human-specific probe.
(B) A summary of the levels of human cell engraftment by percentage of
human DNA in the BM of mice transplanted with ex vivo-cultured CB and
BM CD34+ cells. Cells were cultured for 3 or 6 days
before transplantation with the cytokine combinations as indicated.
Values shown are the mean ± SE from 7 independent experiments, with
an average of 7 mice per group. The differences between SCF + FL + IL6RIL6 chimera and other combinations or noncultured day 0 cells for
both time points were significant (P = .0002).
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Human SRC are maintained for 10 to 14 days in the presence of IL6RIL6
chimera.
To determine if the addition of SCF, FL, and IL6RIL6 chimera could
maintain SRC for longer periods of time in ex vivo cultures, NOD/SCID
mice were transplanted with cells after 10 to 14 days of culture.
Representative blots demonstrate that the level of engraftment in the
BM of transplanted mice was as high at day 11 compared with day 6 of
cultures (Fig 3A).
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| Fig 3.
Quantitative analysis of SRC after 10 to 14 days of ex
vivo cultures. (A) CB CD34+ cells were transplanted into
NOD/SCID mice (105 cells/mouse) from replicate wells. The
cells in the wells were ex vivo-cultured for 6 or 11 days with SCF + FL + IL6RIL6 chimera. At each time point, the content of 1 well
containing initial cells and their expanded progeny was transplanted
into a mouse. DNA was extracted from the BM of transplanted mice 1 month after transplantation and hybridized with a human-specific probe.
(B) A summary of the levels of human engraftment in mice transplanted
with CD34+ cells that were ex vivo-cultured for 10 to 14 days before transplantation. Data shown are from 4 independent
experiments.
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A total of 30 mice transplanted with CD34+ cells cultured
in vitro with SCF, FL, and IL6RIL6 for 10 to 14 days were analyzed. Fourteen of the mice had levels of engraftment between 2% and 10%,
whereas another 14 mice had lower levels of engraftment between 0.1%
and 1% (Fig 3B). Only 2 mice showed no engraftment. For the 28 mice
(93% of the animals) showing engraftment of human cells cultured in
vitro for these prolonged periods of time with SCF, FL, and IL6RIL6,
the mean level of human DNA was 5%. Hence, despite variations from
donor to donor, a significant level of engraftment was maintained in
the majority of mice, even after prolonged culture in vitro.
Multilineage differentiation of human hematopoietic cells engrafted
in NOD/SCID mice with SCF, FL, and IL6RIL6 chimera.
We verified whether human lymphoid progenitor cells developed in the BM
of the NOD/SCID mice engrafted with cells that were cultured with SCF,
FL, and IL6RIL6 chimera. The cells recovered from the murine BM were
double-stained for human-specific CD45 and for the CD19 B-cell marker.
Figure 4A shows the presence of human
CD45+CD19+ cells in the BM of a mouse that was
transplanted with human CD34+ cells after 10 days in vitro
with IL6RIL6 chimera. The BM cells recovered from the engrafted mouse
were further cultured with SCF and IL-15 for an additional 10 days and
stained for the NK cell marker CD56 (Fig 4B). The presence of 63%
CD45+CD56+ cells demonstrates that the human
lymphoid progenitors have the potential to differentiate also into NK
cells. In parallel, BM cells recovered from engrafted mice gave rise to
both erythroid and myeloid colonies (data not shown). Hence,
CD34+ cells maintained 10 days in ex vivo cultures with
SCF, FL, and IL6RIL6 chimera contain engrafting SRC/stem cells that can
give rise to both myeloid and lymphoid cells in the transplanted
animals.
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| Fig 4.
Lymphoid differentiation of SRC ex vivo-cultured with
IL6RIL6 chimera from the marrow of mice transplanted with
CD34+ cells. Recovered BM cells from a highly engrafted
NOD/SCID mouse transplanted with CD34+ cells that were
cultured with SCF + FL + IL6RIL6 chimera for 10 days before
transplantation. One month after transplantation, the murine BM was
harvested. The cells were stained with lineage-specific markers and
with isotype control for detection of nonspecific staining. (A) Murine
BM cells were stained with antihuman CD45-FITC and antihuman CD19-PE
for detection of pre-B cells. (B) BM cells from the transplanted mouse
were further cultured with SCF + IL-15 for 10 days before staining
with human CD45-FITC and antihuman CD56-PE for detection of NK cells.
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The IL6RIL6 chimera preferentially stimulates
CD34+ CD38/low cells.
Whereas nearly all CD34+ cells from human CB were found to
express gp130, there is a small fraction that do not express the IL-6R
(data not shown). This finding is in line with previous reports, which
found that CD34+gp130+IL-6R
cells contain higher levels of more primitive LTC-IC.26
Because these cells lack membranal IL-6R, they require both IL-6 and
sIL-6R to activate gp130 signaling. Previous results have demonstrated that SRC are a very small subpopulation of the CD34+
cells,34 and our data show that, within the total
CD34+ population, SRC can be stimulated by IL6RIL6 chimera
much more efficiently than by a mixture of IL-6 + sIL-6R (Fig 2). We
found that IL6RIL6 chimera binds to gp130 with much higher affinity than the mixture of IL-6 and sIL-6R added separately
(Fig 5). Therefore, because of its higher
affinity, IL6RIL6 chimera could also bind to the rare
gp130+ SRC cell subpopulation, triggering gp130
dimerization and transduction of the IL-6-type biological signal.
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| Fig 5.
Relative affinities of IL6RIL6 chimera and IL-6 + sIL-6R mixture for gp130. Scatchard plot of the dose-dependent binding
of the IL6RIL6 chimera alone ( ) or of free sIL-6R in the presence of
a constant amount of IL-6 ( ). Binding measured on immobilized pure
soluble gp130 as described in Materials and Methods. Calculated
kd were 6 × 1011 mol/L for IL6RIL6
chimera and 2.5 × 1010 mol/L for the sIL-6R + IL-6
mixture.
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To investigate the response of CD34+ subpopulations to
stimulation with these cytokines, we separated the very early
CD34+CD38/low cells that contain SRC
from the more differentiated CD34+CD38+
population. In vitro, 6-day, serum-free cultures showed
(Table 1) that the
CD34+CD38/low cells proliferated only
when IL6RIL6 was added to SCF + FL (6-fold increase in cell number). In
contrast, CD34+CD38+ cells stimulated with SCF + FL together with IL6RIL6 chimera produced only a small 1.5-fold
increase over cells cultured with SCF + FL alone (Table 1). A
similar difference was seen in the number of colony forming
progenitors, which were increased 12.9-fold by IL6RIL6 chimera in the
primitive CD34+CD38/low cell population
and only 1.3-fold in the more mature
CD34+CD38+ cell population compared with
SCF + FL alone (Table 1). In 5 different experiments,
CD34+CD38/low cells were found to
respond to the addition of IL6RIL6 chimera by a large increase of both
total cell numbers and progenitor levels after 6-day cultures
(Fig 6A, I + II). The differences in cell
growth and requirement for IL6RIL6 chimera between total CD34+ cells (Fig 1) and
CD34+CD38/low cells (Table 1 and Fig 6A)
reflect the low percentage of CD38 cells in the
heterogeneous population of CD34+ cells.
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Table 1.
Cell Numbers and Progenitor Levels of
CD34+CD38/low and
CD34+CD38+ Cells Cultured With IL6RIL6
Chimera for 6 Days
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| Fig 6.
Cell numbers, progenitors, and engraftment levels of
CD34+CD38/low cells after ex vivo
cultures. Human CD34+CD38/low cells were
cultured in serum-free media with cytokines as indicated and were
assayed for cell numbers, progenitors, and levels of human cell
engraftment. (A, I) Cell numbers. Mean increase over day 0: SCF + FL
= 1.4, SCF + FL + IL6RIL6 = 6.1 (P = .02). (A, II)
Progenitor levels. Mean increase over day 0: SCF + FL = 1.9, SCF + FL + IL6RIL6 = 12.8 (P = .03). Values shown are the
mean ± SE from 5 independent experiments. (B) Representative Southern
blots of the BM of mice transplanted with ex vivo-cultured
CD34+CD38/low cells that originated from 2 different donors. DNA was extracted from the BM of transplanted mice 1 month after transplantation and hybridized with a human-specific probe.
The marrow of mice transplanted with 104 uncultured cells
before seeding (lane 1) expanded cells cultured for 6 days with SCF + FL either alone (lane 2) or together with IL6RIL6 chimera (lane 3). The
marrow of mice transplanted with 104 ex vivo-cultured for
10 days with SCF + FL either alone (lane 4) or together with IL6RIL6
chimera (lane 5). (C) A summary of the levels of human cell engraftment
by percentage of human DNA in the BM of mice transplanted with ex
vivo-cultured CD34+CD38/low cells.
NOD/SCID mice were transplanted with initial 104
CD34+CD38/low cells that were ex
vivo-cultured for 6 to 10 days in serum-free media with SCF + FL
alone compared with SCF + FL + IL-6 + sIL-6R (P = .04)
or compared with SCF + FL + IL6RIL6 chimera (P = .02).
Values shown are the mean ± SE from 5 independent experiments (n = 17).
|
|
The repopulating potential of ex vivo-cultured
CD34+· CD38/low cells was assayed by
transplantation into NOD/SCID mice. Representative Southern blots of
individual mice show a 10-fold increase in the level of engraftment
when IL6RIL6 chimera was added during the 6-day cultures (Fig 6B, lane
3) compared with SCF + FL alone (lane 2) or with noncultured day 0 cells (lane 1). The same pattern was seen after 10 days of culture
(lane 5 v lane 4). Analysis of all experiments with
CD34+CD38/low cells cultured for 6 to 10 days showed that the mean level of engraftment was increased 19.8-fold
by the addition of IL6RIL6 chimera to SCF + FL compared
with SCF + FL alone (Fig 6C, P = .02). With the
purified CD34+CD38/low cells, a 6.4-fold
increase in the levels of engraftment was also seen when the IL-6 + sIL-6R mixture (instead of IL6RIL6 chimera) was added to SCF + FL (Fig
6C, P = .04). Therefore, as expected, the enrichment achieved
by subfractionation of the CD34+ cells allows the SRC cells
to respond better to the IL-6/sIL-6 receptor-type signal, because they
now represent a higher percentage of the total gp130+ population.
These results were supported by CD34/CD38 phenotype analysis of ex
vivo-cultured cells. When total CD34+ cells were cultured
for 11 days with SCF + FL + IL6RIL6 chimera, the
CD34+CD38/low subpopulation was twice as
high than in cultures with SCF + FL alone (data not shown). Cultures of
sorted CD34+CD38/low cells were analyzed
for maintenance of the primitive cells during prolonged cultures of up
to 14 days. When CD34+CD38/low cells
were cultured with SCF + FL and IL6RIL6 chimera, we found 28.2%,
29.5%, and 9.1% of CD34+CD38/low cells
at days 6, 10, and 14, respectively (data not shown). Previous reports
on cultures with SCF, FL, and IL-6 indicated complete loss of
CD34+CD38/low cells after 9 days.7
Increased human cell engraftment by the IL6RIL6 chimera-treated cells
was also demonstrated by assaying the levels of human clonogenic
progenitors recovered from the BM of transplanted NOD/SCID mice
(Table 2). Human progenitor levels were
increased 16-fold when IL6RIL6 chimera was added to SCF + FL in the ex
vivo cultures. Furthermore, BM from mice engrafted with
CD34+CD38/low cells that had been
cultured with SCF, FL, and IL6RIL6 chimera before transplantation gave
rise to multilineage human progenitors that included myeloid
colony-forming unit-granulocyte-macrophage (CFU-GM) as well
as erythroid burst-forming unit-erythroid (BFU-E) and colony-forming
unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) mixed colonies. Without IL6RIL6, mostly
myeloid CFU-GM were observed (Table 2 and data not shown).
View this table:
[in this window]
[in a new window]
|
Table 2.
Human Progenitors in the BM of NOD/SCID Mice
Transplanted With CD34+CD38/low Cells That
Were Cultured Ex Vivo for 6 Days
|
|
The maintenance activity provided by the IL6RIL6 chimera, together with
the cell expansion, probably accounts for the marked increase in
engraftment levels of the human hematopoietic cells in the transplanted
NOD/SCID mice.
| |
DISCUSSION |
The establishment of the NOD/SCID model enables us to measure the in
vivo engraftment properties of primitive human hematopoietic cells. One
issue of current interest is the potential of ex vivo-cultured and
expanded human hematopoietic cells to repopulate in vivo the BM by
proliferation and multilineage differentiation.
Recently, several groups suggested different cytokine combinations for
maintenance and expansion of primitive human hematopoietic cells in ex
vivo cultures.4-8 The stem cell activities in these cytokine-stimulated cultures were evaluated in vitro by the levels of
colony-forming cells (CFC) or LTC-IC6,38 as
well as by expression of differentiation markers such as CD34, CD38, or
HLA-DR.6,8,38 In vivo models were used in which cultured
cells were transplanted into immunodeficient mice6-8 or
into sheep fetuses in utero.39 In transplanted NOD/SCID
mice, human SRC could be maintained in vitro for 4 days7 or
human CRU for 5 to 8 days6 and still engraft the marrow of
transplanted mice, but successful maintenance of SRC for longer periods
was not reported, apparently due to differentiation and loss of
repopulating stem cell potential.7,34 In the sheep chimera
model, transplantation of cells after ex vivo culture for 14 days
resulted in only short-term engraftment in the marrow of in utero
fetuses, but after the fetuses were born no human engraftment was
detected.39
A combination of IL-6 and sIL-6R together with either SCF24
or FL25 was shown to increase the levels of
CD34+ cells and progenitors in vitro, and we previously
reported that this IL-6 + sIL-6R combination increases progenitors even
when added to cultures containing both SCF and FL.29
Therefore, despite the synergistic effect of SCF with FL,40
IL-6 can further stimulate progenitors when provided with its sIL-6R
agonist, which allows gp130 signaling in very early
CD34+gp130+IL-6R cell
subpopulations.26 We also showed that a fused IL6RIL6 chimeric glycoprotein is very active in increasing colony-forming progenitors when added to SCF + FL,29 and another such
fusion protein named Hyper-IL-6 was shown to increase progenitor
levels when added to SCF + IL-3.28 The higher activity of
proteins in which sIL-6R is fused to IL-6 can be explained, as we
demonstrate here, by a higher affinity of IL6RIL6 chimera to gp130
compared with the IL-6 + sIL-6R mixture (Fig 5).
Our current study shows the effect of the IL6RIL6 chimera added to SCF
and FL for SRC maintenance and expansion in cultures of
CD34+ and CD34+CD38/low
human CB and BM cells. Assayed in the NOD/SCID model, CD34+
cultures containing the IL6RIL6 chimera produced higher levels of
engraftment compared with day 0 noncultured cells or compared with
cells stimulated with SCF + FL either alone or with a mixture of IL-6 + sIL-6R. Total levels of human cell engraftment depend on the purity of
enriched transplanted cells and occasionally fluctuate upon different
donors and mice, resulting in a wide range of engraftment levels in the
marrow of transplanted mice. Hence, despite the relatively low
engraftment levels obtained in some experiments, the described growth
effect of IL6RIL6 chimera on CD34+ and
CD34+CD38/low cells was consistently
observed. Furthermore, in the presence of IL6RIL6 chimera, we could
prolong the period of ex vivo cultures for up to 14 days and still
achieve high levels of human cell engraftment. CD34+ cells
cultured with the IL6RIL6 chimera added to SCF + FL demonstrated multilineage differentiation in the murine BM, generating myeloid, erythroid, and lymphoid progenitor cells, including NK cells. Moreover,
human progenitors recovered from the murine BM produced higher numbers
of colonies, and particularly immature colonies such as CFU-GEMM, when
the mice were transplanted with cells cultured with IL6RIL6 as compared
with SCF + FL alone. Together, these features indicate the maintenance
of stem cell properties in CD34+ cells ex vivo-cultured in
the presence of the IL6RIL6 chimera.
A possible mechanism for IL6RIL6 chimera stimulation may be the
differential expression of IL-6R and of gp130 on the cell surface of
CD34+ cells. In the CD34+ population, all cells
express gp130, but the levels of IL-6R vary and are undetectable in a
small subpopulation of the cells. A previous study demonstrated that
CD34+IL-6R cells are more primitive than
CD34+IL-6R+ cells.26 Because the
IL6RIL6 chimera binds directly with high affinity to isolated gp130, it
is likely that, in the CD34+ population, cells lacking
IL-6R on their surface are still capable of gp130-mediated response to
IL6RIL6 chimera. The more primitive cells may have an enhanced response
to IL6RIL6 chimera stimulation compared with more mature cells. This
was verified by examining the potential of the IL6RIL6 chimera to
induce proliferation and to enhance SRC in very early
CD34+CD38/low subpopulations isolated
from CB CD34+ cells.
Our study shows that IL6RIL6 chimera stimulated mainly the very early
CD34+CD38/low cells, which responded by
extensive proliferation. There was even a higher increase in progenitor
levels than in total cell numbers, supporting the notion that IL6RIL6
chimera is a very early acting cytokine/soluble receptor complex.
Indeed, the more mature CD34+CD38+ cells were
hardly affected, exhibiting only a limited increase in cell numbers and
in progenitors.
Previous studies with SCF, FL, IL-3, granulocyte colony-stimulating
factor (G-CSF), and IL-6 by itself reported a decrease in the
engrafting potential of SRC after 8 days of in vitro culture, accompanied by loss of the early
CD34+CD38 compartment as detected by
flow cytometry.7 In contrast, in this report, flow
cytometry analyses of sorted
CD34+CD38/low cells cultured in the
presence of IL6RIL6 chimera demonstrate maintenance of these early
cells after 6 and 10 days of ex vivo stimulation. Even after 14 days, a
remarkable percentage of CD34+CD38/low
cells were still observed in ex vivo cultures. This maintenance of the
CD34+CD38/low phenotype can account for
the much higher levels of engraftment in the marrow of transplanted
NOD/SCID mice after IL6RIL6 chimera cultures compared with cells
cultured with SCF + FL alone. Engraftment by these cells, as
demonstrated by DNA analysis and recovery of human progenitors from the
murine BM, was higher with IL6RIL6 chimera than with the IL-6 + sIL-6R combination.
These findings demonstrate that the present CHO-produced IL6RIL6
chimera is an important hematopoietic stimulator that supports maintenance as well as proliferation of primitive
CD34+CD38/low cells in ex vivo cultures.
In addition, induction of extensive proliferation of early SRC, while
maintaining their repopulating potential, suggests that the IL6RIL6
chimera may be useful for clinical transplantation protocols.
| |
ACKNOWLEDGMENT |
The assistance of Zipora Marks, Rosalie Kaufman, and Nili Nissin is
gratefully acknowledged. We thank D. Novick and M. Rubinstein for help
in providing anti-IL-6R antibodies.
| |
FOOTNOTES |
Submitted September 9, 1998; accepted March 28, 1999.
Supported in part by grants from the Israel Academy of Science (T.L.),
the Balfur Peisner Bone Marrow Cancer Research Fund (O.K.), the Ares
Serono group (T.L. and M.R.), and MINERVA Foundation, Munich/Germany
(R.A.) and by National Institutes of Health Grant No. A130389 (L.S.).
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.
Address reprint requests to Tsvee Lapidot, PhD, Incumbent of the
Pauline Recanati Career Development Chair of Immunology, Department of
Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel;
e-mail: litsvee{at}weizmann.weizmann.ac.il.
| |
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Blood,
May 15, 2000;
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3102 - 3105.
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L. D. Shultz, P. A. Lang, S. W. Christianson, B. Gott, B. Lyons, S. Umeda, E. Leiter, R. Hesselton, E. J. Wagar, J. H. Leif, et al.
NOD/LtSz-Rag1null Mice: An Immunodeficient and Radioresistant Model for Engraftment of Human Hematolymphoid Cells, HIV Infection, and Adoptive Transfer of NOD Mouse Diabetogenic T Cells
J. Immunol.,
March 1, 2000;
164(5):
2496 - 2507.
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ABSTRACT
INTRODUCTION