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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-09-2782.
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Blood, 15 April 2003, Vol. 101, No. 8, pp. 2924-2931
PLENARY PAPER
SCID-repopulating cell activity of human cord blood-derived
CD34 cells assured by intra-bone marrow
injection
Jianfeng Wang,
Takafumi Kimura,
Rumiko Asada,
Sachio Harada,
Shouhei Yokota,
Yoshio Kawamoto,
Yoshihiro Fujimura,
Takashi Tsuji,
Susumu Ikehara, and
Yoshiaki Sonoda
From the Department of Hygiene, the Department of
Digestive Surgery, and the Third Department of Internal Medicine, Kyoto
Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku,
Kyoto; the Department of Blood Transfusion Medicine, Nara Medical
University, Shijocho, Kashihara, Nara; the Department of Industrial
Science and Technology, Tokyo University of Science, Yamazaki, Noda,
Chiba; and the First Department of Pathology, Transplantation
Center, Kansai Medical University, Fumizonocho, Moriguchi, Osaka,
Japan.
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Abstract |
Precise analysis of human CD34-negative
(CD34 ) hematopoietic stem cells (HSCs)
has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34 severe combined
immunodeficiency (SCID)- repopulating cells (SRCs) with extensive
lymphoid and myeloid repopulating ability using the intra-bone marrow
injection (IBMI) technique. Lineage-negative (Lin)
CD34 cells did not show SRC activity by conventional
tail-vein injection, possibly due to their low levels of homing
receptor expression and poor SDF-1/CXCR4- mediated homing abilities,
while they clearly showed a high SRC activity by IBMI. They generated
CD34+ progenies not only in the injected left tibia but
also in other bones following migration. Moreover, they showed slower
differentiating and reconstituting kinetics than CD34+
cells in vivo. These in vivo-generated CD34+ cells showed
a distinct SRC activity after secondary transplantation, clearly
indicating the long-term human cell repopulating capacity of our
identified CD34 SRCs in nonobese diabetic (NOD)/SCID
mice. The unveiling of this novel class of primitive human
CD34 SRCs by IBMI will provide a new concept of the
hierarchy in the human HSC compartment and has important implications
for clinical HSC transplantation as well as for basic research of HSC.
(Blood. 2003;101:2924-2931)
© 2003 by The American Society of Hematology.
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Introduction |
The most primitive hematopoietic stem cells
(HSCs) in mammals, including mice, rhesus monkeys, and humans, have
long been believed to be CD34 antigen (Ag)-positive
(CD34+).1 In fact, bone marrow (BM) and
peripheral blood stem cell (PBSC) transplantation studies indicate that
a CD34+ subpopulation in the BM or PB can provide durable
long-term donor-derived lymphohematopoietic
reconstitution,2,3 although longer-term observations are
necessary. Therefore, we used CD34 Ag to identify/purify immature
hematopoietic stem/progenitor cells. However, Osawa et al challenged
this long-standing dogma, and their studies have revealed that murine
long-term lymphohematopoietic reconstituting HSCs are lineage marker
negative (Lin)
c-kit+Sca-1+CD34-low/negative
(CD34lo/).4 From another point of view,
Goodell et al have reported that a unique class of HSCs (side
population [SP] cells) expressing low or undetectable levels of CD34
Ag exists in multiple species, including mice, rhesus monkeys, and
humans, using the fluorescent DNA-binding dye, Hoechst
33342.5 They speculated that human SP cells have long-term
repopulating ability, as do murine SP cells. Collectively, these
studies imply the existence of a hitherto unidentified population of
primitive human HSCs that lack the CD34 Ag expression.
One of the assay systems that can measure the repopulation and
differentiation capacities of human HSCs is the SCID-repopulating cell
(SRC) assay developed by Dick and his colleagues.6-8 Using this system, Bhatia et al first reported that SRCs were present in
human BM- and cord blood (CB)-derived
LinCD34 cells.9 Their
multilineage reconstituting analyses of CD34-negative (CD34) SRCs clearly demonstrated that the vast majority
of CD45+ human cells in murine BMs were CD19+ B
cells. Also, limiting dilution analysis indicated that there was one
SRC in 125 000 LinCD34 cells. On the other
hand, the frequency of CD34 SRCs increased to 1 in
38 000 cells after 4 days of short-term culture of these
LinCD34 cells in the presence of a cocktail
of cytokines or human umbilical vein endothelial cell-conditioned
medium. They suggested that unidentified cells, termed "pre-SRCs,"
present in the CD34 cell population, might acquire some
homing molecules necessary for redistribution to nonobese
diabetic/severe combined immunodeficiency (NOD/SCID) mouse BM after
tail-vein injection (TVI).
The existence of long-term repopulating CD34 HSCs in
human BM-derived Lin cells also is supported by the
reported data, in which the CD34 fraction of normal human
BM contains cells capable of engraftment and differentiation into
CD34+ progenitors as well as multiple lymphohematopoietic
lineages using the human/sheep competitive engraft
model.10 However, studies on human CD34 HSCs
have been hindered by the lack of a positive marker, comparable to the
Sca-1 in mice. In this study, we try to further characterize the
CD34 SRCs present in the human CB and further unveil the
unidentified pre-SRCs using the intra- BM injection (IBMI)
technique.11 Our analysis of the proliferation and
differentiation capacities of purified CB-derived
LinCD34 cells, both in vivo and in vitro,
demonstrate the existence of a distinct class of HSCs with extensive
lymphoid and myeloid differentiation capacity as well as secondary
repopulating ability in NOD/SCID mice. This novel class of
CD34 SRCs detected by IBMI may correspond to the
temporarily termed pre-SRCs.9
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Materials and methods |
Collection of CB samples and processing
CB samples were obtained from normal full-term deliveries with
signed informed consent and approved by the Institutional Review Board
of Kyoto Prefectural University of Medicine. The cells were processed
within 24 hours of collection. Mononuclear cells (MNCs) were isolated
using Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden) density
gradient centrifugation. MNCs were further enriched by negative
depletion of 8 lineage-positive cells, including CD3, CD14, CD16, CD19,
CD24, CD56, CD66b, and glycophorin A (GPA) using a StemSep device
(StemCell Technologies, Vancouver, BC, Canada), as
reported.12
Purification of LinCD34 cells
The above-mentioned semipurified cells (Figure 1A) were stained
with 13 fluorescein isothiocyanate (FITC)-conjugated lineage-specific mAbs against CD2, CD16, CD24, GPA (all from DAKO, Kyoto, Japan), CD3,
CD19 (both from eBioscience, San Diego, CA), CD4, CD10, CD20, CD41 (all
from Beckman Coulter, Fullerton, CA), CD7, CD56 (both from Nichirei,
Tokyo, Japan), CD14 (Becton Dickinson, San Jose, CA), phycoerythrin
(PE)-conjugated anti-CD34 mAb (Becton Dickinson), and PC5-conjugated
anti-CD45 mAb (Beckman Coulter). Availability of these
lineage-specific mAbs used for cell sorting was confirmed beforehand.
Then 13 lineage-positive cells remaining in the immunomagnetically separated cells were further gated out (Figure 1B). These
Lin cells were sorted into CD34high,
CD34low, and CD34 cells (Figure 1D) using a
FACSVantage (Becton Dickinson) as reported.12,13 Approximately 20% to 40% of the CD34 cell fraction in
the immunomagnetically separated cells was recovered in the sorted
LinCD34 cell fraction (R5 gate in
Figure 1D).
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| Figure 1.
Characterization of purified CB-derived
LinCD34 cells.
(A) The forward scatter/side scatter (FSC/SSC) profile of
immunomagnetically separated cells. The R1 gate was set on the
lymphocyte window. (B) Cell-surface expression of 13 lineage markers,
including CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41,
CD56, and GPA on cells residing in the R1 gate. Cells residing in the
R2 gate were further subdivided into 3 fractions according to their
expression levels of CD34 antigen. (C) Isotype control. (D) Cells
residing in the R3, R4, and R5 gates were classified as
LinCD34high,
LinCD34low, and
LinCD34 cells, respectively. The
definitions of CD34high, CD34low, and
CD34 fractions are as follows: the CD34high
fraction contains cells expressing maximum phycoerythrin (PE)
fluorescent intensity (FI) to 15% level of FI; the CD34low
fraction contains cells expressing 5% to 1% level of FI; and the
CD34 fraction contains cells expressing less than 0.5%
level of FI, respectively. (E-G) The expression patterns of CD34
antigen on CD45+ cells derived from the 7-day cocultures of
CD34high (E), CD34low (F), and
CD34 (G) cells with the murine stromal cell HESS-5 in the
presence of a cocktail of cytokines.
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RT-PCR analysis for CD34 mRNA
Total RNA was isolated from sorted cells and cell lines (KG1 and
Daudi for positive and negative controls, respectively) using RNeasy
Mini Kit (QIAGEN GmbH, Hilden, Germany) and transcribed to
complementary DNA with avian myeloblastosis virus (AMV) reverse transcriptase (Life Science, St Petersburg, FL). Oligonucleotide primers for human CD34 were synthesized: 5'-CTAGCCTTGCAACATCTCCC-3' (sense), 5'-GAATAGCTCTGGTGGCTTGC-3' (antisense), resulting in a PCR
product of 409 bp. PCR amplification (35 cycles of 30 seconds at
94°C, 45 seconds at 57°C, and 60 seconds at 72°C) was conducted on a block thermal cycler (Perkin-Elmer, Norwalk, CT). Reverse transcription-polymerase chain reaction (RT-PCR) products from all samples were electrophoresed on a 2.0% agarose gel.
Analysis of expression pattern of CXCR4 and other adhesion
molecules on LinCD34high,
CD34low, and CD34 cells by flow
cytometry
The immunomagnetically separated cells were stained with the
above-mentioned FITC-conjugated 13 lineage-specific mAbs,
allophycocyanin (APC)-conjugated anti-CD34 mAb (DAKO),
PerCP-conjugated anti-CD45 mAb (Becton Dickinson), PE-conjugated
anti-CD62L mAb (Beckman Coulter), and biotinylated mAbs for CXCR4
(Genzyme Techne, Minneapolis, MN), CD31 and CD49d (both from Ancell,
Bayport, MN), and CD54 and CD106 (both from eBioscience). After
washing, the cells were incubated with streptavidin-PE (Becton
Dickinson). All flow cytometric analyses were done on a FACSCalibur
(Becton Dickinson), as reported.12,13
Clonal cell culture and coculture with HESS-5 cells
Human colony-forming cells (CFCs) were assayed using our
standard methylcellulose cultures as reported.12-14 Sorted
LinCD34high, CD34low,
CD34 cells were plated at 1 × 104 cells
per 6-well plate onto pre-established irradiated HESS-515 layers in StemPro-34 medium (Gibco Laboratories, Grand Island, NY) and
a cocktail of recombinant human cytokines, including 300 ng/mL stem
cell factor (SCF), 300 ng/mL flt3 ligand (FL), 300 ng/mL thrombopoietin
(TPO), 10 ng/mL interleukin-3 (IL-3), 10 U/mL IL-6, and 10 ng/mL
granulocyte (G)-colony stimulating factor (CSF), and 5% fetal calf
serum (FCS, Hyclone Laboratories, Logan, UT).
IBMI of purified cells
Intra-BM injection (IBMI) was carried out as reported previously
with modifications.11 Briefly, after sterilization of the skin around the left knee joint, the knee was flexed to 90 degrees, and
the proximal side of the tibia was drawn to the anterior. A 27-gauge
needle was inserted into the joint surface of the tibia through the
patellar tendon and then inserted into the BM cavity. Using a
Hamilton microsyringe, the specified number of donor cells per
10 µL of  -medium were carefully injected from the bone hole into
the BM cavity.
SCID-repopulating cell (SRC) assay
An SRC assay was performed using the methods reported
previously,7,8 with modifications. Five-week-old
NOD/Shi-scid/scid (NOD/SCID) mice were obtained
from the Central Institute for Experimental Animals (Kawasaki, Japan).
The animal experiments were approved by the Animal Care Committee of
Kyoto Prefectural University of Medicine. All mice were handled in
sterile conditions and maintained in germ-free isolators located in the
Central Laboratory Animal Facility. In this study, purified
5 × 104 CB-derived
LinCD34high,
LinCD34low, or
LinCD34 cells were transplanted by TVI or
IBMI into sublethally irradiated (250 cGy using a 137Cs-
irradiator) 8- to 12-week-old mice. NOD/SCID mice receiving transplants
of 5 × 104 LinCD34high cells
showed equivalently high repopulation efficiencies compared with those
for mice receiving transplants of more than 1 × 105
LinCD34high cells by TVI or IBMI (data not
shown). In some experiments, 5 × 103
LinCD34high cells were transplanted by IBMI.
The mice were killed 5 to 16 weeks after transplantation, and the BMs
from the pairs of femurs, tibiae, and humeri of each mouse were flushed
into -medium containing 10% FCS. To assess the frequency of SRCs in
the CB-derived LinCD34high and
LinCD34 cells, NOD/SCID mice received
transplants of various doses of LinCD34high
cells (range, 300 to 1250 cells, n = 26) and
LinCD34 cells (range, 5000 to 40 000
cells, n = 21) by IBMI. After 12 weeks, the rates of human
CD45+ cells in the murine BMs were analyzed by flow
cytometry. Mice were scored as positive if more than 0.1% of total
murine BM cells were human CD45+. The frequencies of SRCs
were calculated using Poisson statistics as
reported.16
Analysis of human cell engraftment in NOD/SCID mice by
flow cytometry
The repopulation of human hematopoietic cells in murine BMs was
determined by detecting the number of cells positively stained with
PC5-conjugated anti-human CD45 mAb (Beckman Coulter). The cells also
were stained with PE-conjugated anti-human CD34 mAb (Becton Dickinson)
and FITC-conjugated mAbs for human lineage-specific Ags, including
CD14 (Becton Dickinson), CD19 (eBioscience), CD33 and CD41 (both from
Beckman Coulter), and GPA (DAKO) for the detection of specific subsets
of human hematopoietic cells. Briefly, BM cells were suspended in
Ca2+- and Mg2+-free phosphate-buffered saline
(PBS) containing 2% FCS after lysis of red blood
cells. The cells then were incubated with human immunoglobulin G
(IgG), followed by staining with the above-mentioned mAbs.
First, the R1 gate was set on the total BM cells (Figure 4). Human
hematopoietic subsets, except for GPA, were quantified by gating on
human CD45+ cells (Figure 4, R2 gate) and then assessing
those stained with anti-human CD34 and various mAbs for
lineage-specific Ags. GPA+ cells were quantified in whole
BM cells (no gate) without lysis of red blood cells.
Transwell migration assay
To assess CXCR4-mediated transmigration of CD34+
SRCs and IBMI-CD34 SRCs in vitro, a total of
1 × 105 immunomagnetically separated
cells12 were allowed to migrate toward a gradient of SDF-1
as previously reported.17,18 Briefly, 125 ng/mL of rh
SDF-1 (Genzyme/Techne, Cambridge, MA) was added to the lower chamber
of a Costar 24-well transwell (Corning, NY) containing X-VIVO 20 (Biowhittaker, Walkersville, MD) supplemented with 0.5% BSA. The
transwell inserts (5.0-µm pore size, Corning) were placed, and the
above-mentioned cells were then inoculated into the upper chamber.
After 4 hours of incubation at 37°C with 5% CO2, both
migrating cells in the lower chamber and nonmigrating cells in the
upper chamber were recovered. The LinCD34high
and LinCD34 cells were then sorted from the
migrating and nonmigrating fractions using a FACSVantage as described.
The respective 5 × 103 migrating and nonmigrating
LinCD34high cells and 5 × 104
migrating and nonmigrating LinCD34 cells
were transplanted by IBMI or TVI into irradiated recipient mice as
described. After 12 weeks, the repopulation of human CD45+
cells in murine BMs was determined by flow cytometry.
Secondary transplantation
For secondary transplantations, murine BM cells were obtained
from the pairs of femurs, tibiae, and humeri of highly engrafted primary recipient mice 8 to 16 weeks after transplantation with 5 × 104 LinCD34high or 12 to
16 weeks after transplantation with 5 × 104
LinCD34 cells by IBMI. The human cell
repopulation rates in the primary recipients' BMs for
CD34+ SRCs and IBMI-CD34 SRCs were 31% to
80% and 15% to 40%, respectively. Whole BM cells were stained with
PE-conjugated anti-CD34 mAb (Becton Dickinson) and PC5-conjugated
anti-CD45 mAb (Beckman Coulter). The human CD45+CD34+ and
CD45+CD34 cells then were sorted using a
FACSVantage (Becton Dickinson). These sorted CD34+ or
CD34 cells were transplanted by IBMI into irradiated
secondary recipient mice. Twelve weeks after transplantation, the
presence of human CD45+ cells in the secondary recipients'
BMs was analyzed by flow cytometry, as described for primary transplantation.
Statistical analysis
The significance of differences was determined using the
Mann-Whitney U test.
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Results |
Characterization of purified CB-derived
LinCD34 cells
We first depleted the lineage-positive cells from CB-derived
mononuclear cells using an immunomagnetic system.12 These
cells were further labeled with a mixture of 13 monoclonal antibodies (mAbs) (Figure 1B, R2 gate) and then were
subdivided into 3 distinct populations based on their surface CD34 Ag
expression (Figure 1D). We sorted these 3 fractions. The phenotypic
purity of the sorted cells consistently exceeded 99% when checked
using postsorting flow cytometric analysis.
Contamination of the LinCD34 cell fraction
with CD34+ cells was ruled out by a semiquantitative RT-PCR
(data not shown).
The colony-forming capacities of these 3 fractions were quite
different. The LinCD34high fraction contained
approximately 50% to 60% myeloid, 40% to 50% erythroid, and 2% to
10% mixed CFCs. However, the vast majority (more than 95%) of CFCs in
the LinCD34low fraction were erythroid
progenitors. The LinCD34 fraction showed
almost no colony formation. To characterize these 3 fractions in more
detail, we analyzed the expression patterns of CD38 and CD95 (Fas) by
flow cytometry. Interestingly, the proportion of CD38
cells in the LinCD34low population was only
4.1%, which is significantly lower than that in the other 2 populations (16.9% for LinCD34high and
50.0% for LinCD34 cells). These results
are consistent with the reported data8,16 that
CD34+ SRCs were highly enriched in a
LinCD34+CD38 cell population.
Moreover, the expression rate of CD95 antigen on
LinCD34low cells (19.5%) was much higher
than that in the other 2 populations (2.6% for
LinCD34high and 1.8% for
LinCD34 cells). These results strongly
indicate that the LinCD34low cell population
contains more committed progenitors (mostly erythroid burst-forming cells).
Next, we tested the SRC activity of our 3 purified fractions of cells
by conventional TVI. All 13 mice that received transplants of
LinCD34high cells were engrafted with human
cells. The level of human CD45+ cells in murine BMs was
3.0% to 70.8% (median, 26.2%). In contrast, neither the 9 mice that
received transplants of LinCD34low cells nor
the 10 mice that received transplants of
LinCD34 cells were engrafted with human
cells (Figure 2A-C, left columns).
Phenotypic and functional characterizations of these 3 fractions were
further determined by the cocultures of these cells with the murine
stromal cell line HESS-515,19 and in the presence of SCF,
FL, TPO, IL-3, IL-6, and G-CSF. After a 7-day coculture of
LinCD34high and
LinCD34 cells with HESS-5, significant
numbers of CD34+ cells were identified (Figure 1E,G). When
1 × 104 LinCD34high cells were
cocultured with HESS-5 in the presence of a cocktail of cytokines for 7 days, approximately 3.9 × 106 human CD45+
cells (390 folds) were recovered, and 30% of them were still CD34+ cells. In the case of the 1 × 104
LinCD34 cells, approximately
1.7 × 105 human CD45+ cells (17 folds) were recovered, and 18% of them turned out to be
CD34+. On the other hand, the flow cytometric pattern for
LinCD34low cells (Figure 1F) was very
different from the other 2 patterns observed for
LinCD34high and
LinCD34 cells. Namely,
1 × 104 LinCD34low cells
yielded 1 × 106 human CD45+ cells (100 folds). However, only 1.6% of them were CD34+
after the coculture. We then transplanted these 3 fractions of cells
recovered from the cocultures into respective 5 NOD/SCID mice using
TVI. The levels of human cell engraftment were respectively 31% to
81% (median, 34.0%) for cultured
LinCD34high cells and 0.3% to 12% (median,
5.7%) for cultured LinCD34 cells. These
results indicate that the cultured LinCD34
cell fraction contained the SRCs, which could not home into the BM
niche by TVI before the coculture. However, none of the 5 mice that
received transplants of cultured LinCD34low
cells were engrafted with human cells.
These results clearly imply that the
LinCD34 population is a distinct population
and differs from both LinCD34high and
LinCD34low cells, not only in terms of CD34,
CD38, and CD95 expression, but also in terms of the proliferation
kinetics, colony-forming ability, and SRC activity.
SRC activity of CB-derived LinCD34high,
CD34low, or CD34 cells using the intra-bone
marrow injection
Lapidot and his colleagues clearly demonstrated that the chemokine
stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a
pivotal role in the homing and repopulation of CD34+ SRCs
in NOD/SCID mice.17,18 Very recently, it was reported that
CXCR4, VLA-4, and VLA-5 played important roles in the homing of
CD34+ SRCs by TVI as well as IBMI.20 Moreover,
the homing of HSCs to the BM can be considered as a multistep process
in which various adhesion molecules present both on HSCs and BM
endothelial cells are involved.21-23 Accordingly, we
analyzed the expression patterns of CXCR4 and other adhesion molecules
on the surfaces of CB-derived LinCD34high,
LinCD34low, or
LinCD34 cells by flow cytometry.
Significant numbers of CB-derived LinCD34high
cells expressed CXCR4, CD31, CD49d, CD54, CD62L, and CD106. However, LinCD34 cells expressed lower levels of
CXCR4, CD62L, and CD106 (data not shown). In addition, the low level of
surface CXCR4 expression on CB-derived
LinCD34CD38 cells has been
reported previously, as has their poor SDF-1-induced migration and
undetectable homing potential in murine BM and spleen.18 Therefore, we hypothesized that very primitive repopulating HSCs that
lack the CD34 Ag expression may not home into the BM niche by TVI,
since LinCD34 cells expressed the low
levels of these homing receptors. Thus, we used the IBMI
technique11 and tested the SRC activity of these 3 fractions of cells.
When LinCD34high cells were transplanted
using IBMI, all 9 mice were repopulated, and the level of human cell
engraftment was 12.8% to 80.0% (median, 64.8%) (Figure 2A, right
column). Very interestingly, this repopulating rate was significantly
higher than that by conventional TVI (P < .03). Next, we
transplanted LinCD34 cells using IBMI.
Surprisingly, all 7 mice were repopulated, and the level of human cell
engraftment was 10.0% to 52.6% (median, 19.3%) (Figure 2C, right
column). On the other hand, none of the 7 mice that received
transplants of LinCD34low cells using IBMI
were engrafted with human cells (Figure 2B, right column). These
results clearly indicate that the CB-derived LinCD34 cell population contains SRCs
detected only by IBMI, which we have called
IBMI-CD34 SRCs.
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| Figure 2.
Human CD45+ cell engraftment of NOD/SCID
mice.
Each mouse that received a transplant of 5 × 104
CB-derived LinCD34high (A),
LinCD34low (B), and
LinCD34 (C) cells was killed 12 weeks after
transplantation. Open and closed circles represent the repopulation
rates in total murine BMs by conventional TVI and by IBMI,
respectively. Horizontal bars represent each median of the repopulation
rates. The level of repopulation by
LinCD34high cells by IBMI (median, 64.8%) is
significantly (P < .03) higher than that (median, 26.2%)
by TVI. All 7 mice that received transplants of
LinCD34 cells by IBMI were engrafted, and
the median human CD45+ cell rate is 19.3%, while none of
the 10 mice that received transplants of
LinCD34 cells by TVI were engrafted. In
addition, none of the mice that received transplants of
LinCD34low cells by TVI or IBMI were
engrafted.
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In the above-mentioned mice that received transplants either with
LinCD34 or
LinCD34high cells by IBMI, we separately
analyzed the human cell repopulation in the injected left tibiae and
the other bones, including right tibia and pairs of femur and humerus
(Figure 3). In these representative mice
that received transplants of CD34+ SRCs or
IBMI-CD34 SRCs, the human CD45+ cells were
clearly detected not only in the injected left tibia but also in the
other bones. In addition, significant numbers of CD34+
progenies were generated at both sites. These results indicate that
IBMI-CD34 SRCs as well as CD34+ SRCs could
migrate from the injected site to the other bones.
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| Figure 3.
In vivo generation of human CD34+ cells.
In this study, LinCD34high or
LinCD34 cells (5 × 104) were
transplanted into the left tibia of NOD/SCID mice using the IBMI
technique. After 12 weeks, the rates of CD45+ and
CD34+ cells in the injected left tibiae and other bones
(right tibia + 2 femurs + 2 humeri) were separately analyzed
by flow cytometry. In these representative mice that received
transplants of CD34+ SRCs and IBMI-CD34 SRCs,
both showed the marked repopulation with human CD45+ cells.
Importantly, both SRCs generated the significant numbers of
CD34+ cells not only at the site of injection, but also at
the destinations of migration. The numbers in the quadrants define
percentages of these cells.
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Comparison of differentiation potentials of CB-derived
IBMI-CD34 SRCs and CD34+ SRCs
To further evaluate the functional differences between
CD34 and CD34+ SRCs, we studied their
multilineage reconstitution abilities using IBMI. In our SRC assay
system, all NOD/SCID mice that received transplants either of
5 × 103 LinCD34high cells or
5 × 104 LinCD34 cells by
IBMI showed signs of human cell engraftment. Limiting dilution analysis
demonstrated that the frequencies of repopulating cells in CB-derived
LinCD34high and
LinCD34 cells were 1/1010 and 1/24 100,
respectively. These results imply that 5 × 103
LinCD34high cells or 5 × 104
LinCD34 cells contain approximately 4 or 5 and 2 or 3 SRCs, respectively. Analysis of the 2 representative mice
that received transplants either of
LinCD34high cells (Figure 4, left
column) or
LinCD34 cells (Figure 4, right column)
clearly indicate that both the CD34+ SRCs and
IBMI-CD34 SRCs have an extensive differentiation
potential to B-lymphoid, myeloid, monocytic, megakaryocytic, and
erythroid lineages in vivo.
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| Figure 4.
The multilineage reconstitution abilities of
CD34+ SRCs and IBMI-CD34 SRCs.
First, the R1 gate was set on the total murine BM cells obtained from
these representative mice 12 weeks after the transplantation of
5 × 103 LinCD34high or
5 × 104 LinCD34 cells, and
then human CD45+ cells were gated as R2. Expression of
lineage markers, including CD19 (pan-B cell), CD33 (myeloid), CD14
(monocytic), and CD41 (megakaryocytic) on the R2-gated cells was
analyzed by 3-color flow cytometry. Only the expression of GPA
(erythroid) was analyzed on the whole BM cells (no gate). In this
particular mouse that received a transplant of CD34+ SRCs
(left column), 39.2% of total BM cells were human CD45+
cells, which contained 11.0% of CD34+ cells, 50.1% of
CD19+ cells, 5.7% of CD33+ cells, 5.3% of
CD14+ cells, and 2.4% of CD41+ cells. In
addition, 1.4% of whole murine BM cells were human GPA+
cells. In another representative mouse that received a transplant of
IBMI-CD34 SRCs (right column), 52.4% of total BM cells
were human CD45+ cells, which contained 13.0% of
CD34+ cells, 19.1% of CD19+ cells, 4.0% of
CD33+ cells, 3.2% of CD14+ cells, and 1.9% of
CD41+ cells. In addition, 0.5% of whole murine BM cells
were human GPA+ cells.
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Next, the percentages of lineage-positive cells expressing CD19, CD33,
CD14, CD41, and GPA were compared (Figure
5). These results demonstrated that
CD34+ SRCs could supply more mature lymphohematopoietic
cells at 12 weeks after transplantation than did
IBMI-CD34 SRCs, which showed slow differentiation
kinetics. However, the percentages of CD34+ cells were
comparable in both SRCs (Figure 5). CD34+ SRCs transplanted
by TVI showed almost comparable multilineage reconstitution potential
(data not shown).
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| Figure 5.
Comparison of the differentiation potential of
IBMI-CD34 SRCs with that of CD34+SRCs.
The multilineage reconstitution abilities of IBMI-CD34
(gray columns) and CD34+ (open columns) SRCs using IBMI
technique were compared. Each bar represents the median of positive
rates obtained from 3 mice that received transplants of either
5 × 103 LinCD34high or
5 × 104 LinCD34 cells. In
human CD45+ cells, the rate of CD34+ cells was
almost comparable in both populations. On the other hand, the rates of
CD19+, CD33+, CD14+, and
CD41+ in human CD45+ cells and GPA+
cells in whole BM cells were significantly (P < .05)
higher in the mice that received transplants of CD34+ SRCs
than in those that received IBMI-CD34 SRCs.
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Kinetics of engraftment potential of CB-derived
IBMI-CD34 SRCs: a comparison with CD34+
SRCs
As a next approach to characterize the IBMI-CD34
SRCs with respect to repopulating potential, we analyzed the kinetics
of engraftment following IBMI of purified
LinCD34 cells and compared the repopulating
pattern with that of LinCD34high cells
(Figure 6). In this experiment, both mice
that received transplants of LinCD34 and
LinCD34high cells showed signs of human cell
repopulation at 5 weeks after transplantation. At 8 weeks, the
percentage of human CD45+ cells in mice that received
transplants of LinCD34high cells markedly
increased to 16.1% (median), which is significantly (P < .05) higher than at 5 weeks (median, 2.9%). At 12 weeks, the percentage of human CD45+ cells for
CD34+ SRCs (median, 30.5%) was maintained at the same
level. In contrast, that (median, 4%) for IBMI-CD34 SRCs
at 8 weeks was comparable to the level of human cell repopulation at 5 weeks, while it significantly (P < .05) increased to
37.1% (median) at 12 weeks.
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| Figure 6.
Kinetics of repopulation of NOD/SCID mice by
IBMI-CD34 and CD34+ SRCs.
In this study, a group of 18 mice (8 weeks old) received transplants of
5 × 103 LinCD34high or
5 × 104 LinCD34 cells
isolated from 6 CB samples. Groups of 3 mice were killed at 5, 8, and
12 weeks after transplantation. At each time point, BM cells obtained
from pairs of femurs, tibiae, and humeri were analyzed by flow
cytometry for their contents of human CD45+ cells. Open and
gray columns represent the values of CD45+ cells derived
from CD34+ SRCs and IBMI-CD34 SRCs,
respectively. Horizontal bars represent the respective medians. Both
CD34+ SRCs and IBMI-CD34 SRCs showed signs of
engraftment at 5 weeks after transplantation. The human
CD45+ cell rate for CD34+ SRCs significantly
(P < .05) increased from 5 weeks to 8 weeks, while that
for IBMI-CD34 SRCs significantly (P < .05)
increased from 8 weeks to 12 weeks.
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These results indicated that IBMI-CD34 SRCs show delayed
or slow reconstitution kinetics when transplanted into NOD/SCID mice using IBMI and suggested that IBMI-CD34 SRCs are in a
more profoundly dormant state than CD34+ SRCs.
SDF-1/CXCR4-mediated migration ability of IBMI-CD34
SRCs and CD34+ SRCs
To assess migration ability toward a gradient of SDF-1 of
IBMI-CD34 SRCs as well as CD34+ SRCs, we
performed a transwell migration assay. Results are presented in Table
1. As expected, migrating
LinCD34high cells repopulated all 5 NOD/SCID
mice both by TVI and IBMI. Interestingly, nonmigrating
LinCD34high cells also showed distinct SRC
activity only by IBMI. These results suggest that the CB-derived
LinCD34high cell population contains at least
2 types of SRCs. Our identified nonmigrating
IBMI-CD34+SRCs may represent the
CD34+CXCR4 SRCs, recently reported by Kollet
et al.24 These unique SRCs express intracellular
CXCR4, which can be functionally expressed on the cell membrane to
mediate SDF-1-induced homing and repopulation. In the case of
LinCD34 cells, the migrating cells did not
show any SRC activity by IBMI. Surprisingly, nonmigrating
LinCD34 cells did repopulate all 3 mice by
IBMI. These results demonstrate that the IBMI is much more sensitive
than TVI for detecting both CD34 and CD34+
SRCs, which have poor SDF-1/CXCR4-mediated migration ability.
Secondary repopulating ability of IBMI-CD34 SRCs and
CD34+ SRCs
To further evaluate the long-term repopulating potential of
IBMI-CD34 as well as CD34+ SRCs, BM cells
obtained from each primary recipient mice were assessed for their SRC
activity by secondary transplantation. First, we transplanted
LinCD34high cells to primary mice by IBMI.
After 8 to 16 weeks, we serially transplanted sorted human
CD45+CD34+ and
CD45+CD34 cells obtained from primary
recipient mouse BMs by IBMI. As presented in Table
2, only CD34+ cells could
repopulate approximately 70% (11 of 15) of secondary recipient mice.
The human CD45+ cell rate in these mice was 0.1% to 11.0%
(median, 4.7%). On the other hand, none of the secondary mice that
received transplants of sorted CD34 cells were engrafted
with human cells. These results indicate that human CD34+
SRCs do not convert to CD34 SRCs for at least 16 weeks
after transplantation.
In the case of primary mice receiving transplants of
LinCD34 cells by IBMI, all 5 mice were
highly engrafted with human CD45+ cells (15% to 40%). Of
note was that sorted CD34+ cells repopulated 80% (4 of 5)
of the secondary recipient mice, and their human CD45+ cell
rates were 0.1% to 0.9% (median, 0.15%). Interestingly, none of the
sorted CD34 cells engrafted secondary recipients. These
results clearly indicate that IBMI-CD34 SRCs have the
capacity to generate CD34+ SRCs in vivo as well as
long-term human cell repopulating capacity in NOD/SCID mice.
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Discussion |
A number of studies concerning murine and human CD34
primitive HSCs have suggested that the CD34lo/ cell
population contains long-term lymphohematopoietic repopulating HSCs.4,5,9,10,19 However, precise analysis of human
CD34 HSCs has been hindered by the lack of a simple and
reliable assay system of these rare cells. In this study, we
established a dependable assay system for CD34 SRCs using
the IBMI technique.
As described, IBMI-CD34 SRCs cannot home into the BM
niche by TVI. This is partly explained by their lower expression levels of homing receptors, including CXCR4. The transwell migration assay
toward a gradient of SDF-1 clearly indicated that
IBMI-CD34 SRCs have poor SDF-1/CXCR4-mediated migration
and homing abilities. An analysis of the in vivo migration ability of
HSCs (Figure 3) demonstrated that a significant proportion of
IBMI-CD34 SRCs as well as CD34+ SRCs were
redistributed from the injected left tibia to the other bones and
proliferated at the migrated sites, where both SRCs generated
significant numbers of CD34+ progenies. However, it remains
unknown whether the IBMI-CD34 SRCs migrate to the other
bones with the CD34 immunophenotype or after their
conversion to the CD34+ state. Furthermore, the molecular
mechanisms involved in this migratory (redistribution and homing)
process are yet to be clarified.
Secondary transplantation studies of sorted
CD45+CD34+ and
CD45+CD34 cells obtained from primary
recipient mice that received transplants either of
IBMI-CD34 SRCs or CD34+ SRCs demonstrated
that only CD34+ cells could repopulate secondary recipient
mice. These results indicated that IBMI-CD34 SRCs
generated CD34+ SRCs in vivo and are consistent with
reported data that human CB-derived LinCD34
cells generated a large number of CD34+ stem cells in an ex
vivo culture system using HESS-5 and various human
cytokines.25 More importantly, the secondary
transplantation studies demonstrated for the first time that CB-derived
IBMI-CD34 SRCs have long-term (up to 28 weeks) human cell
repopulating capacity in NOD/SCID mice.
In contrast to murine BM-derived HSCs,26 human CB-derived
CD34+ SRCs did not convert to CD34 SRCs for
at least 16 weeks after transplantation. However, it will require a
longer period of observation (more than 1 year) to elucidate the
possibility of reversion of CD34 antigen expressed on human CB-derived
CD34+ HSCs, as suggested by Zanjani et al using human
BM-derived HSCs.27 In addition, it was not clarified
whether the CD34 cell population obtained from primary
mice that received transplants of LinCD34 |
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Abstract
Introduction