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

Precise analysis of human CD34-negative(CD34ⴚ) hematopoietic stem cells (HSCs)
has been hindered by the lack of a simple
and reliable assay system of these rarecells. Here, we successfully identify human cord blood–derived CD34ⴚ severe
combined immunodeficiency (SCID)–
repopulating cells (SRCs) with extensive
lymphoid and myeloid repopulating ability using the intra–bone marrow injection(IBMI) technique. Lineage-negative (Linⴚ)
CD34ⴚ 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 theyclearly showed a high SRC activity by
IBMI. They generated CD34ⴙ progenies
not only in the injected left tibia but also
in other bones following migration. Moreover, they showed slower differentiating
and reconstituting kinetics than CD34ⴙ
cells in vivo. These in vivo–generated
CD34ⴙ cells showed a distinct SRC activity after secondary transplantation, clearly
indicating the long-term human cell re-

populating capacity of our identified
CD34ⴚ SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel
class of primitive human CD34ⴚ SRCs by
IBMI will provide a new concept of thehierarchy in the human HSC compartment and has important implications forclinical HSC transplantation as well as forbasic research of HSC. (Blood. 2003;101:2924-2931)© 2003 by The American Society of Hematology

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, bonemarrow (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 theexistence 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 anddifferentiation capacities of human HSCs is the SCID-repopulatingcell (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 Lin⫺CD34⫺ cells.9 Their
multilineage reconstituting analyses of CD34-negative (CD34⫺)
SRCs clearly demonstrated that the vast majority of CD45⫹ humancells in murine BMs were CD19⫹ B cells. Also, limiting dilution
analysis indicated that there was one SRC in 125 000 Lin⫺CD34⫺
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
Lin⫺CD34⫺ cells in the presence of a cocktail of cytokines orhuman umbilical vein endothelial cell–conditioned medium. Theysuggested 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 inhuman 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 apositive marker, comparable to the Sca-1 in mice. In this study, we
try to further characterize the CD34⫺ SRCs present in the human

From the Department of Hygiene, the Department of Digestive Surgery, and theThird 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; theDepartment 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.

Supported in part by a Grant-in-Aid for Scientific Research B from the Ministryof Education, Science and Culture of Japan.

Submitted September 11, 2002; accepted December 2, 2002. Prepublished online as
Blood First Edition Paper, December 12, 2002; DOI 10.1182/blood-2002-09-2782.

2924

Reprints: Yoshiaki Sonoda, the Department of Hygiene, Kyoto Prefectural
University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602-8566,
Japan; e-mail: sonoda@basic.kpu-m.ac.jp.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

CB and further unveil the unidentified pre-SRCs using the intraBM injection (IBMI) technique.11 Our analysis of the proliferationand differentiation capacities of purified CB-derived Lin⫺CD34⫺ 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

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 depletionof 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 LinⴚCD34ⴚ 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 (allfrom 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 lineagespecific 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 wasrecovered in the sorted Lin⫺CD34⫺ cell fraction (R5 gate in Figure 1D).
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 weresynthesized: 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 LinⴚCD34high, CD34low, and CD34ⴚ cellsby flow cytometry
The immunomagnetically separated cells were stained with the abovementioned 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

SCID-REPOPULATING ACTIVITY OF CD34⫺ CELLS BY IBMI

2925

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 Lin⫺CD34high, CD34low,
CD34⫺ cells were plated at 1 ⫻ 104 cells per 6-well plate onto preestablished irradiated HESS-515 layers in StemPro-34 medium (Gibco
Laboratories, Grand Island, NY) and a cocktail of recombinant humancytokines, 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 withmodifications.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 ␮L 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,7,8with 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 werehandled in sterile conditions and maintained in germ-free isolators locatedin the Central Laboratory Animal Facility. In this study, purified 5 ⫻ 104
CB-derived Lin⫺CD34high, Lin⫺CD34low, or Lin⫺CD34⫺ 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 transplantsof 5 ⫻ 104 Lin⫺CD34high cells showed equivalently high repopulation
efficiencies compared with those for mice receiving transplants of morethan 1 ⫻ 105 Lin⫺CD34high cells by TVI or IBMI (data not shown). In someexperiments, 5 ⫻ 103 Lin⫺CD34high cells were transplanted by IBMI. The
mice were killed 5 to 16 weeks after transplantation, and the BMs from thepairs 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 Lin⫺CD34high and Lin⫺CD34⫺ cells, NOD/SCID mice received transplants of various doses of Lin⫺CD34high cells (range, 300 to
1250 cells, n ⫽ 26) and Lin⫺CD34⫺ cells (range, 5000 to 40 000 cells,n ⫽ 21) by IBMI. After 12 weeks, the rates of human CD45⫹ cells in themurine 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 asreported.16
Analysis of human cell engraftment in NOD/SCID miceby flow cytometry
The repopulation of human hematopoietic cells in murine BMs wasdetermined 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 (bothfrom Beckman Coulter), and GPA (DAKO) for the detection of specificsubsets 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 humanimmunoglobulin G (IgG), followed by staining with the above-mentioned

2926

WANG et al

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

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 humanCD45⫹ cells (Figure 4, R2 gate) and then assessing those stained withanti–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 IBMICD34⫺ 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-␮m 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 theupper chamber were recovered. The Lin⫺CD34high and Lin⫺CD34⫺ cells
were then sorted from the migrating and nonmigrating fractions using a
FACSVantage as described. The respective 5 ⫻ 103 migrating and nonmigrating Lin⫺CD34high cells and 5 ⫻ 104 migrating and nonmigrating
Lin⫺CD34⫺ cells were transplanted by IBMI or TVI into irradiatedrecipient mice as described. After 12 weeks, the repopulation of humanCD45⫹ cells in murine BMs was determined by flow cytometry.
Secondary transplantation
For secondary transplantations, murine BM cells were obtained from thepairs of femurs, tibiae, and humeri of highly engrafted primary recipient
mice 8 to 16 weeks after transplantation with 5 ⫻ 104 Lin⫺CD34high or 12
to 16 weeks after transplantation with 5 ⫻ 104 Lin⫺CD34⫺ 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⫹ orCD34⫺ 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.

Results
Characterization of purified CB-derived LinⴚCD34ⴚ 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 ofthe sorted cells consistently exceeded 99% when checked using
postsorting flow cytometric analysis.
Contamination of the Lin⫺CD34⫺ 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 Lin⫺CD34high 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
Lin⫺CD34low fraction were erythroid progenitors. The Lin⫺CD34⫺
fraction showed almost no colony formation. To characterize these

Figure 1. Characterization of purified CB-derived LinⴚCD34ⴚ 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 R5gates were classified as Lin⫺CD34high, Lin⫺CD34low, and Lin⫺CD34⫺ cells, respectively. The definitions of CD34high, CD34low, and CD34⫺ fractions are as follows: theCD34high fraction contains cells expressing maximum phycoerythrin (PE) fluorescentintensity (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% levelof 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) cellswith the murine stromal cell HESS-5 in the presence of a cocktail of cytokines.

3 fractions in more detail, we analyzed the expression patterns of
CD38 and CD95 (Fas) by flow cytometry. Interestingly, theproportion of CD38⫺ cells in the Lin⫺CD34low population was only
4.1%, which is significantly lower than that in the other 2
populations (16.9% for Lin⫺CD34high and 50.0% for Lin⫺CD34⫺
cells). These results are consistent with the reported data8,16 that
CD34⫹ SRCs were highly enriched in a Lin⫺CD34⫹CD38⫺ cell
population. Moreover, the expression rate of CD95 antigen on
Lin⫺CD34low cells (19.5%) was much higher than that in the other2 populations (2.6% for Lin⫺CD34high and 1.8% for Lin⫺CD34⫺
cells). These results strongly indicate that the Lin⫺CD34low cell
population contains more committed progenitors (mostly erythroid
burst-forming cells).
Next, we tested the SRC activity of our 3 purified fractions ofcells by conventional TVI. All 13 mice that received transplants of
Lin⫺CD34high cells were engrafted with human cells. The level ofhuman CD45⫹ cells in murine BMs was 3.0% to 70.8% (median,
26.2%). In contrast, neither the 9 mice that received transplants of
Lin⫺CD34low cells nor the 10 mice that received transplants of
Lin⫺CD34⫺ 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 themurine 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
Lin⫺CD34high and Lin⫺CD34⫺ cells with HESS-5, significant
numbers of CD34⫹ cells were identified (Figure 1E,G). When1 ⫻ 104 Lin⫺CD34high cells were cocultured with HESS-5 in thepresence 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
Lin⫺CD34⫺ cells, approximately 1.7 ⫻ 105 human CD45⫹ cells(17 folds) were recovered, and 18% of them turned out to be

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

Figure 2. Human CD45ⴙ cell engraftment of NOD/SCID mice. Each mouse thatreceived a transplant of 5 ⫻ 104 CB-derived Lin⫺CD34high (A), Lin⫺CD34low (B), and
Lin⫺CD34⫺ (C) cells was killed 12 weeks after transplantation. Open and closed
circles represent the repopulation rates in total murine BMs by conventional TVI andby IBMI, respectively. Horizontal bars represent each median of the repopulation
rates. The level of repopulation by Lin⫺CD34high cells by IBMI (median, 64.8%) issignificantly (P ⬍ .03) higher than that (median, 26.2%) by TVI. All 7 mice thatreceived transplants of Lin⫺CD34⫺ cells by IBMI were engrafted, and the medianhuman CD45⫹ cell rate is 19.3%, while none of the 10 mice that received transplantsof Lin⫺CD34⫺ cells by TVI were engrafted. In addition, none of the mice that received
transplants of Lin⫺CD34low cells by TVI or IBMI were engrafted.

CD34⫹. On the other hand, the flow cytometric pattern for
Lin⫺CD34low cells (Figure 1F) was very different from the other 2
patterns observed for Lin⫺CD34high and Lin⫺CD34⫺ cells. Namely,1 ⫻ 104 Lin⫺CD34low 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 recoveredfrom the cocultures into respective 5 NOD/SCID mice using TVI.
The levels of human cell engraftment were respectively 31% to81% (median, 34.0%) for cultured Lin⫺CD34high cells and 0.3% to12% (median, 5.7%) for cultured Lin⫺CD34⫺ cells. These results
indicate that the cultured Lin⫺CD34⫺ cell fraction contained theSRCs, which could not home into the BM niche by TVI before thecoculture. However, none of the 5 mice that received transplants ofcultured Lin⫺CD34low cells were engrafted with human cells.
These results clearly imply that the Lin⫺CD34⫺ population is adistinct population and differs from both Lin⫺CD34high andLin⫺CD34low cells, not only in terms of CD34, CD38, and CD95
expression, but also in terms of the proliferation kinetics, colonyforming ability, and SRC activity.

SCID-REPOPULATING ACTIVITY OF CD34⫺ CELLS BY IBMI

2927

CD62L, and CD106. However, Lin⫺CD34⫺ cells expressed lower
levels of CXCR4, CD62L, and CD106 (data not shown). In
addition, the low level of surface CXCR4 expression on CBderived Lin⫺CD34⫺CD38⫺ 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 hypothesizedthat very primitive repopulating HSCs that lack the CD34 Ag
expression may not home into the BM niche by TVI, since
Lin⫺CD34⫺ 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 Lin⫺CD34high 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
Lin⫺CD34⫺ cells using IBMI. Surprisingly, all 7 mice wererepopulated, and the level of human cell engraftment was 10.0% to52.6% (median, 19.3%) (Figure 2C, right column). On the other
hand, none of the 7 mice that received transplants of Lin⫺CD34low
cells using IBMI were engrafted with human cells (Figure 2B, rightcolumn). These results clearly indicate that the CB-derived
Lin⫺CD34⫺ cell population contains SRCs detected only by IBMI,
which we have called IBMI-CD34⫺ SRCs.
In the above-mentioned mice that received transplants eitherwith Lin⫺CD34⫺ or Lin⫺CD34high cells by IBMI, we separately
analyzed the human cell repopulation in the injected left tibiae andthe other bones, including right tibia and pairs of femur andhumerus (Figure 3). In these representative mice that received
transplants of CD34⫹ SRCs or IBMI-CD34⫺ SRCs, the humanCD45⫹ 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.

SRC activity of CB-derived LinⴚCD34high, CD34low, or CD34ⴚ
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⫹ SRCsin NOD/SCID mice.17,18 Very recently, it was reported thatCXCR4, VLA-4, and VLA-5 played important roles in the homingof CD34⫹ SRCs by TVI as well as IBMI.20 Moreover, the homingof HSCs to the BM can be considered as a multistep process inwhich various adhesion molecules present both on HSCs and BM
endothelial cells are involved.21-23 Accordingly, we analyzed theexpression patterns of CXCR4 and other adhesion molecules on thesurfaces of CB-derived Lin⫺CD34high, Lin⫺CD34low, or Lin⫺CD34⫺
cells by flow cytometry. Significant numbers of CB-derived
Lin⫺CD34high cells expressed CXCR4, CD31, CD49d, CD54,

Figure 3. In vivo generation of human CD34ⴙ cells. In this study, Lin⫺CD34high or
Lin⫺CD34⫺ cells (5 ⫻ 104) were transplanted into the left tibia of NOD/SCID miceusing the IBMI technique. After 12 weeks, the rates of CD45⫹ and CD34⫹ cells in theinjected 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 withhuman 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.

2928

WANG et al

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

Comparison of differentiation potentials of CB-derived
IBMI-CD34ⴚ SRCs and CD34ⴙ SRCs

To further evaluate the functional differences between CD34⫺ andCD34⫹ SRCs, we studied their multilineage reconstitution abilities
using IBMI. In our SRC assay system, all NOD/SCID mice thatreceived transplants either of 5 ⫻ 103 Lin⫺CD34high cells or
5 ⫻ 104 Lin⫺CD34⫺ cells by IBMI showed signs of human cellengraftment. Limiting dilution analysis demonstrated that thefrequencies of repopulating cells in CB-derived Lin⫺CD34high andLin⫺CD34⫺ cells were 1/1010 and 1/24 100, respectively. These
results imply that 5 ⫻ 103 Lin⫺CD34high cells or 5 ⫻ 104
Lin⫺CD34⫺ cells contain approximately 4 or 5 and 2 or 3 SRCs,
respectively. Analysis of the 2 representative mice that received
Figure 5. Comparison of the differentiation potential of IBMI-CD34ⴚ SRCs withthat of CD34ⴙSRCs. The multilineage reconstitution abilities of IBMI-CD34⫺ (graycolumns) 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 Lin⫺CD34high or 5 ⫻ 104 Lin⫺CD34⫺ cells. In humanCD45⫹ 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 micethat received transplants of CD34⫹ SRCs than in those that received
IBMI-CD34⫺ SRCs.

transplants either of Lin⫺CD34high cells (Figure 4, left column) or
Lin⫺CD34⫺ 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.
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 moremature lymphohematopoietic cells at 12 weeks after transplantation than did IBMI-CD34⫺ SRCs, which showed slow differentiation kinetics. However, the percentages of CD34⫹ cells werecomparable in both SRCs (Figure 5). CD34⫹ SRCs transplanted by
TVI showed almost comparable multilineage reconstitution potential (data not shown).

Figure 4. The multilineage reconstitution abilities of CD34ⴙ SRCs and IBMICD34ⴚ SRCs. First, the R1 gate was set on the total murine BM cells obtained fromthese representative mice 12 weeks after the transplantation of 5 ⫻ 103 Lin⫺CD34highor 5 ⫻ 104 Lin⫺CD34⫺ 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 thewhole 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, whichcontained 11.0% of CD34⫹ cells, 50.1% of CD19⫹ cells, 5.7% of CD33⫹ cells, 5.3% ofCD14⫹ 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 transplantof 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 wholemurine BM cells were human GPA⫹ cells.

Figure 6. Kinetics of repopulation of NOD/SCID mice by IBMI-CD34ⴚ and CD34ⴙ
SRCs. In this study, a group of 18 mice (8 weeks old) received transplants of 5 ⫻ 103
Lin⫺CD34high or 5 ⫻ 104 Lin⫺CD34⫺ 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⫹ SRCsand 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.

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

SCID-REPOPULATING ACTIVITY OF CD34⫺ CELLS BY IBMI

Table 1. SDF-1–induced migration ability of CD34ⴙ and IBMI-CD34ⴚ SRCs
Transplanted cells

Transplantationroute

Incidence ofengraftment

Human CD45⫹
cells (%)*

0.7-15.2 (5.5)

Lin⫺CD34highMigrating

IBMI

5/5

Migrating

TVI

5/5

0.1-2.4 (0.3)

Nonmigrating

IBMI

5/5

0.8-12.5 (2.5)

Nonmigrating

TVI

1/5

0.1

Lin⫺CD34⫺
Migrating

IBMI

0/3

0

Nonmigrating

IBMI

3/3

0.5-3.5 (3.1)

Immunomagnetically separated cells were allowed to migrate toward a gradientof SDF-1 as described in “Materials and methods.” After the transwell migration, bothmigrating and nonmigrating cells were recovered. These cells were sorted into
Lin⫺CD34high and Lin⫺CD34⫺ cells. Cells (5 ⫻ 103 Lin⫺CD34high and 5 ⫻ 104
Lin⫺CD34⫺) were then transplanted into NOD/SCID mice by IBMI or TVI, as
indicated. After 12 weeks, repopulation of human CD45⫹ cells in murine BMs wasanalyzed.
*Median values are presented in parentheses.

Kinetics of engraftment potential of CB-derived IBMI-CD34ⴚ
SRCs: a comparison with CD34ⴙ SRCs

As a next approach to characterize the IBMI-CD34⫺ SRCs withrespect to repopulating potential, we analyzed the kinetics ofengraftment following IBMI of purified Lin⫺CD34⫺ cells andcompared the repopulating pattern with that of Lin⫺CD34high cells(Figure 6). In this experiment, both mice that received transplantsof Lin⫺CD34⫺ and Lin⫺CD34high cells showed signs of human cell
repopulation at 5 weeks after transplantation. At 8 weeks, thepercentage of human CD45⫹ cells in mice that received transplantsof Lin⫺CD34high 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.
These results indicated that IBMI-CD34⫺ SRCs show delayedor slow reconstitution kinetics when transplanted into NOD/SCID
mice using IBMI and suggested that IBMI-CD34⫺ SRCs are in amore profoundly dormant state than CD34⫹ SRCs.

2929

Lin⫺CD34high cells also showed distinct SRC activity only by
IBMI. These results suggest that the CB-derived Lin⫺CD34high 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 thecell membrane to mediate SDF-1–induced homing and repopulation. In the case of Lin⫺CD34⫺ cells, the migrating cells did notshow any SRC activity by IBMI. Surprisingly, nonmigrating
Lin⫺CD34⫺ 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-CD34ⴚ SRCs andCD34ⴙ SRCs

To further evaluate the long-term repopulating potential of IBMICD34⫺ as well as CD34⫹ SRCs, BM cells obtained from eachprimary recipient mice were assessed for their SRC activity bysecondary transplantation. First, we transplanted Lin⫺CD34highcells to primary mice by IBMI. After 8 to 16 weeks, we seriallytransplanted 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 humanCD45⫹ 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 toCD34⫺ SRCs for at least 16 weeks after transplantation.
In the case of primary mice receiving transplants of Lin⫺CD34⫺
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.

SDF-1/CXCR4–mediated migration ability of IBMI-CD34ⴚ SRCs
and CD34ⴙ SRCs

Discussion

To assess migration ability toward a gradient of SDF-1 of
IBMI-CD34⫺ SRCs as well as CD34⫹ SRCs, we performed atranswell migration assay. Results are presented in Table 1. As
expected, migrating Lin⫺CD34high cells repopulated all 5 NOD/SCID mice both by TVI and IBMI. Interestingly, nonmigrating

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

Table 2. Secondary repopulating ability of CD34ⴙ and IBMI-CD34ⴚ SRCs
Type and no. of cells
transplanted into PR*% Human cells
in PR†

Sorted cells transplanted
into SR

Incidence ofengraftment in SR% Human cells
in SR‡

Lin⫺CD34high, 5 ⫻ 104

31.4-80.0 (72.1)§

CD45⫹CD34⫹

11/15

0.1-11 (4.7)§

CD45⫹CD34⫺

0/15

0

CD45⫹CD34⫹

4/5

0.1-0.9 (0.15)§

CD45⫹CD34⫺

0/5

0Lin⫺CD34⫺, 5 ⫻ 104

15.3-39.6 (23.4)§*5 ⫻ 104 Lin⫺CD34high cells were estimated to contain 40 to 50 SRCs. In contrast, 5 ⫻ 104 Lin⫺CD34⫺ cells contained only 2 or 3 SRCs.
†Human cell repopulation of BMs in primary recipients (PR) was serially analyzed 8 to 16 weeks after transplantation with Lin⫺CD34high cells and 12 to 16 weeks after
transplantation with Lin⫺CD34⫺ cells.
‡Human cell repopulation of BMs in secondary recipients (SR) was analyzed 12 weeks after secondary transplantation.
§Median values are presented in parentheses.

2930

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

WANG et al

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 transwellmigration 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 wereredistributed from the injected left tibia to the other bones andproliferated 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 otherbones 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⫹ andCD45⫹CD34⫺ cells obtained from primary recipient mice thatreceived 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 Lin⫺CD34⫺ 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, thesecondary transplantation studies demonstrated for the first timethat 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 ofreversion of CD34 antigen expressed on human CB-derived
CD34⫹ HSCs, as suggested by Zanjani et al using humanBM-derived HSCs.27 In addition, it was not clarified whether the
CD34⫺ cell population obtained from primary mice that received
transplants of Lin⫺CD34⫺ cells still contained CD34⫺ HSCs,
which cannot home into the BM niche in secondary recipients evenby IBMI. Further studies will be required to clarify thisimportant issue.
Very recently, Guenechea et al have clearly demonstrated thatthe human cell repopulation in NOD/SCID mice that received
transplants of Lin⫺ CB cells is generally oligoclonal, with exten-

sive variability in the life span and proliferative capacity of
individual SRCs.28 Analysis of unique retroviral integration sites of
the SRCs revealed the existence of different clones of SRCs withvariable self-renewal potential and short-term and long-term repopulating capacity. Therefore, it is important to clarify the functional
heterogeneity of CD34⫹ and CD34⫺ SRCs to understand thehierarchy of human HSCs.29
Functional studies, including analyses of multilineage reconstituting ability, the kinetics of engraftment, the productivity of
CD34⫹ progenies, and secondary repopulating ability revealed thatthese IBMI-CD34⫺ SRCs have HSC characteristics different from
CD34⫹ SRCs8,17,18 and also the previously reported CD34⫺ SRCs.9
All these results imply that our identified IBMI-CD34⫺ SRCs are anovel class of primitive repopulating HSCs that can be detected
only by the sensitive IBMI technique.
The application of this IBMI technique may make it possible todiscover other hitherto unidentified HSCs in various organs or tofind new markers for HSCs. There is also the important question ofwhether the BM or mobilized PB contains an equivalent class of
CD34⫺ SRCs. Moreover, IBMI provides a more efficient engraftment of CD34⫹ SRCs, which can contribute to the early phase of
BM reconstitution.10 In addition, our identified nonmigrating
CD34⫹ SRCs can efficiently home into the BM niche by IBMI.
Therefore, it seems to be imperative that these CD34⫺ SRCs shouldbe transplanted with migrating and nonmigrating CD34⫹ SRCs inclinical HSC transplantation using the IBMI technique.
The majority of cord blood stem cell transplantations (CBSCTs)
have been carried out in children, due to limited numbers of HSCs
in a single CB sample.30 Therefore, successfully engrafting adults
on a routine basis using the IBMI technique would greatly expand
the clinical applicability of CBSCT. We anticipate that the use of
the IBMI technique will have a great impact on clinical transplantation in the near future.

Acknowledgments
The authors are grateful to Dr H. Asano of the School of Nursing,
Kyoto Prefectural University of Medicine, for his critical advice on
statistical analysis; and to Dr M. Kita of the Central Laboratory
Animal Facility for his kind cooperation regarding animal care. Drs
T. Abe, M. Nakao, and H. Fujiki of Kyoto Prefectural University of
Medicine are also acknowledged for their encouragement. The
authors also thank Kirin Brewery (Tokyo, Japan) for providing thevarious growth factors used in this study.

References
1. Krause DS, Fackler MJ, Civin CI, May WS. CD34:
structure, biology, and clinical utility. Blood. 1996;
87:1-13.
2. Vogel W, Scheding S, Kanz L, Brugger W. Clinicalapplications of CD34⫹ peripheral blood progenitor cells (PBPC). Stem Cells. 2000;18:87-92.
3. Civin C, Trischmann T, Kadan NS, et al. Highly
purified CD34-positive cells reconstitute hematopoiesis. J Clin Oncol. 1996;14:2224-2233.
4. Osawa M, Hanada K, Hamada H, Nakauchi H.
Long-term lymphohematopoietic reconstitution bya single CD34-low/negative hematopoietic cell.
Science. 1996;273:242-245.
5. Goodell MA, Rosenzweig M, Kim H, et al. Dyeefflux studies suggest that hematopoietic stem
cells expressing low or undetectable levels of
CD34 antigen exist in multiple species. Nat Med.
1997;3:1337-1345.
6. Dick JE. Normal and luekemic human stem cells

assayed in SCID mice. Semin Immunol. 1996;8:
197-206.

eage expression that includes giving rise toCD34⫹ cells. Exp Hematol. 1998;26:353-360.

7. Vormoor J, Lapidot T, Pflumio F, et al. Immature
human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood. 1994;83:2489-2497.

11. Kushida T, Inaba M, Hisha H, et al. Intra-bonemarrow injection of allogeneic bone marrow cells:
a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice.
Blood. 2001;97:3292-3299.

8. Larochelle A, Vormoor J, Hanenberg H, et al.
Identification of primitive human hematopoietic
cells capable of repopulating NOD/SCID mousebone marrow: implications for gene therapy. Nat
Med. 1996;2:1329-1337.
9. Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE.
A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat
Med. 1998;4:1038-1045.
10. Zanjani ED, Almeida-Porada G, Livingston AG,
Flake AW, Ogawa M. Human bone marrow
CD34⫺ cells engraft in vivo and undergo multilin-

12. Minamiguchi H, Yahata N, Kimura T, et al. Interleukin 6 receptor expression by human cord
blood- or peripheral blood-derived primitive
haematopoietic progenitors implies acquisition ofdifferent functional properties. Br J Haematol.
2000;110:327-338.
13. Kimura T, Sakabe H, Tanimukai S, et al. Simultaneous activation of signals through gp130, c-kit,
and interleukin-3 receptor promotes a trilineage
blood cell production in the absence of terminallyacting lineage-specific factors. Blood. 1997;90:4767-4778.

BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8

14. Sonoda Y, Sakabe H, Ohmisono Y, et al. Synergistic actions of stem cell factor and other burstpromoting activities on proliferation of CD34⫹
highly purified blood progenitors expressing
HLA-DR or different levels of c-kit protein. Blood.
1994;84:4099-4106.
15. Tsuji T, Ogasawara H, Aoki Y, Tsurumaki Y, Kodama H. Characterization of murine stromal cellclones established from bone marrow andspleen. Leukemia. 1996;10:803-812.
16. Bhatia M, Wang JCY, Kapp U, et al. Purification ofprimitive human hematopoietic cells capable ofrepopulating immune-deficient mice. Proc Natl
Acad Sci U S A. 1997;94:5320-5325.
17. Peled A, Petit I, Kollet O, et al. Dependence ofhuman stem cell engraftment and repopulation of
NOD/SCID mice on CXCR4. Science. 1999;283:
845-848.
18. Kollet O, Spiegel A, Peled A, et al. Rapid and efficient homing of human CD34⫹CD38⫺/lowCXCR4⫹
stem and progenitor cells to the bone marrow andspleen of NOD/SCID and NOD/SCID/B2mnull
mice. Blood. 2001;97:3283-3291.
19. Nakamura Y, Ando K, Chargui J, et al. Ex vivo
generation of CD34⫹ cells from CD34⫺ hematopoietic cells. Blood. 1999;94:4053-4059.

SCID-REPOPULATING ACTIVITY OF CD34⫺ CELLS BY IBMI

20. Yahata T, Ando K, Sato T, et al. A highly sensitive
strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic
cells into NOD/SCID mice bone marrow. Blood.
2003;101:2905-2913.
21. Papayannopoulou T, Priestley GV, Nakamoto B.
Anti-VLA4/VCAM-1-induced mobilization requires
cooperative signaling through the kit/kit ligand
pathway. Blood. 1998;91:2231-2239.
22. Voermans C, Rood PML, Hordijk PL, Gerritsen
WR, Schoot CE. Adhesion molecules involved intransendothelial migration of human hematopoietic progenitor cells. Stem Cells. 2000;18:435443.
23. Peled A, Kollet O, Ponomaryov T, et al. The chemokine SDF-1 activates the integrins LFA-1,
VLA-4, and VLA-5 on immature human CD34⫹
cells: role in transendothelial/stromal migrationand engraftment of NOD/SCID mice. Blood.
2000;95:3289-3296.
24. Kollet O, Petit I, Kahn J, et al. Human
CD34⫹CXCR4- sorted cells harbor intracellular
CXCR4, which can be functionally expressed andprovide NOD/SCID repopulation. Blood. 2002;
100:2778-2786.
25. Ando K, Nakamura Y, Chargui J, et al. Exten-

2931

sive generation of human cord blood CD34(⫹)
stem cells from Lin(-)CD34(-) cells in a longterm in vitro system. Exp Hematol. 2000;28:
690-699.
26. Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells.
Blood. 1999;94:2548-2554.
27. Zanjani ED, Almeida-Porada G, Livingston AG, etal. Reversible expression of CD34 by adult human bone marrow long-term engrafting cells invivo [abstract]. Blood. 2001;98:72.
28. Guenechea G, Gan OI, Dorrell C, Dick JE. Distinct classes of human stem cells that differ inproliferative and self-renewal potential. Nat Immunol. 2001;2:75-82.
29. Hogan CJ, Shpall EJ, Keller G. Differential longterm and multilineage engraftment potential from
subfractions of human CD34⫹ cord blood cells
transplanted into NOD/SCID mice. Proc Natl
Acad Sci U S A. 2002;99:413-418.
30. Gluckman E, Rocha V, Boyer-Chammard A, et al.
Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med. 1997;
337:373-381.