Leukemia (2010) 24, 162–168& 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00
www.nature.com/leu

ORIGINAL ARTICLE
In vivo dynamics of human cord blood-derived CD34 SCID-repopulating cells using
intra-bone marrow injection
T Kimura1, Y Matsuoka1, M Murakami1,2, T Kimura1,2, M Takahashi1,3, T Nakamoto4, K Yasuda4, K Matsui5, K Kobayashi6,
S Imai6, H Asano7, R Nakatsuka1, Y Uemura1, Y Sasaki1 and Y Sonoda1
1

Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University,
Moriguchi, Osaka, Japan; 2First Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan;
3
Department of Pediatrics, Kansai Medical University, Moriguchi, Osaka, Japan; 4Department of Obstetrics and Gynecology,
Kansai Medical University, Moriguchi, Osaka, Japan; 5Department of Gynecology and Obstetrics, Fukuda Hospital, Kumamoto,Kumamoto, Japan; 6Department of Obstetrics and Gynecology, Aizenbashi Hospital, Osaka, Japan and 7School of Nursing, Kyoto
Prefectural University of Medicine, Kyoto, Japan

The identification of human CD34-negative (CD34) hematopoietic stem cells (HSCs) provides a new concept for thehierarchy in the human HSC compartment. This study investigated the long-term repopulating capacity and redistribution
kinetics of human cord blood-derived CD34 severe combined
immunodeficiency (SCID)-repopulating cells (SRCs) andcompared them with those of CD34 þ CD38 þ and CD34 þ CD38
SRCs using the intra-bone marrow injection (IBMI) to clarify thecharacteristics of CD34 SRCs. On the basis of the limiting
dilution analyses data, estimated numbers of CD34 þ CD38 þ ,CD34 þ CD38, and CD34 SRCs were transplanted to NOD/SCID
mice by IBMI. The human cell repopulation at the site of
injection and the other bones were serially investigated.
Interestingly, CD34 þ CD38 þ , CD34 þ CD38, and CD34 SRCs
began to migrate to other bones 2 and 5 weeks after thetransplantation, respectively. Accordingly, the initiation of
migration seemed to differ between the CD34 þ and CD34
SRCs. In addition, CD34 þ CD38 þ SRCs only sustained a shortterm repopulation. However, both CD34 þ CD38 and CD34
SRCs had longer-term repopulation capacity. Taken together,
these findings showed that CD34 SRCs show different in vivo
kinetics, thus suggesting that the identified CD34 SRCs are a
distinct class of primitive HSCs in comparison to theCD34 þ CD38 þ and CD34 þ CD38 SRCs.
Leukemia (2010) 24, 162–168; doi:10.1038/leu.2009.206;
published online 1 October 2009
Keywords: SCID-repopulating cell; IBMI; CD34; CD38;
hematopoietic stem cell; cord blood

repopulating ability using the intra-bone marrow injection(IBMI) method.7 Functional studies revealed that these CD34
SRCs have different HSC characteristics from earlier-reported
CD34 þ SRCs.7–9 These data suggested that the CD34 SRCs area novel class of primitive repopulating HSCs that can only bedetected by the sensitive IBMI method.7–10
On the other hand, Hogan et al.11 clearly showed that
CB-derived SRCs present in the CD34 þ cell fraction could besegregated into two subpopulations with distinct repopulation
characteristics. The CD34 þ CD38 þ SRCs could rapidly repopulate the recipient NOD/SCID mice, but they could only
maintain a human cell repopulation for 12 weeks, while
showing no secondary repopulating potential. In contrast, themore primitive CD34 þ CD38 SRCs could repopulate recipients
more gradually and maintain human hematopoiesis for at least
20 weeks. Importantly, they had a secondary repopulating
potential throughout the engraftment period. These results
showed that human CB-derived CD34 þ CD38 SRCs are moreprimitive SRCs than CD34 þ CD38 þ SRCs.
To further clarify the HSC characteristics of CD34 SRCs, thisstudy investigated the in vivo redistribution kinetics and thelong-term (LT) repopulating potential of human CB-derived
CD34 SRCs and compared them with those of CD34 þ CD38 þ
and CD34 þ CD38 SRCs using the IBMI method.

Materials and methods
Introduction
The biology of hematopoietic stem cell (HSC) is a current topic
of interest, which has important implications for clinical HSC
transplantation as well as for the basic research of HSCs. A
number of studies have revealed that the severe combined
immunodeficiency (SCID)-repopulating cell (SRC) activity residein CD34-positive (CD34 þ ) cell factions derived from bonemarrow (BM), cord blood (CB), and peripheral blood.1–6
Recently, we successfully identified human CB-derived CD34negative (CD34) SRCs with extensive lymphoid and myeloid
Correspondence: Professor Y Sonoda, Department of Stem Cell
Biology and Regenerative Medicine, Graduate School of Medical
Science, Kansai Medical University, 10-15 Fumizonocho, Moriguchi,
Osaka 570-8506, Japan.
E-mail: sonoda@takii.kmu.ac.jp
Received 25 June 2009; revised 2 August 2009; accepted 24 August
2009; published online 1 October 2009

Collection of CB samples and processing of
lineage-negative cells
CB samples were obtained from normal full-term deliveries withthe signed informed consent. This study was approved by theInstitutional Review Boards of Kansai Medical University. The
CB-derived lineage-negative (Lin) mononuclear cells wereseparated using a StemSep device (StemCell Technologies,
Vancouver, BC, Canada), as reported earlier.7–9

Purification of LinCD34 þ CD38 þ ,LinCD34 þ CD38, and LinCD34 cells

The pooled Lin cells from multiple donors were stained withfluorescein isothiocyanate-conjugated anti-CD45 monoclonal
antibody (mAb) (Beckman Coulter, Inc., Fullerton, CA, USA),
PC5-conjugated anti-CD34 mAb (Coulter), and phycoerythrinconjugated anti-CD38 mAb (Becton Dickinson Immunocytometry, San Jose, CA, USA), as reported earlier.7–9 These stained
cells were then sorted into three fractions, includingIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al

163

Figure 1 Representative FACS profile of sorted CB-derived LinCD45 þ CD34 and LinCD45 þ CD34 þ CD38 þ or LinCD45 þ CD34 þ CD38–
cells. (a) The forward scatter/side scatter (FSC/SSC) profile of immunomagnetically isolated Lin cells. The R1 gate was set on the lymphocyte/blast
window. (b) The R1-gated cells were subdivided into two fractions: LinCD45 þ CD34 þ (R2) and CD45 þ CD34 (R3) cells, respectively. The
definitions of CD34 þ and CD34 cells are as follows: the CD34 þ fraction contains cells expressing maximum PC-5 fluorescent intensity (FI) to 5%
level of FI and the CD34 fraction contains cells expressing o0.2% level of FI, respectively. (c) CD34 þ (R2) cells were further subdivided into two
populations according to their expression levels of the CD38 antigen. The cells in the R4 and R5 gates were classified as CD34 þ CD38 þ andCD34 þ CD38 cells, respectively. The definitions of CD34 þ CD38 þ and CD34 þ CD38 cells are as follows: the CD34 þ CD38 þ fraction contains
cells expressing maximum phycoerythrin FI to 5% level of FI and the CD34 þ CD38 fraction contains cells expressing o0.5% level of FI,
respectively.

LinCD34 þ CD38 þ , LinCD34 þ CD38, and LinCD34 cells
as shown in Figure 1 using a FACSVantage (BD Biosciences,
San Jose, CA, USA).

Semiquantitative reverse transcriptase polymerase chain
reaction analysis for CD34 mRNA
The detailed methods are presented in the legend to
Supplementary Figure 1.

detecting the number of cells positively stained with
PC5-conjugated anti-human CD45 mAb (Coulter) by flow
cytometry (FACS Calibur, Becton Dickinson). The mice werescored as positive if over 0.1% of the total murine BM cells were
human CD45 þ . The cells were also stained with phycoerythrinconjugated anti-human CD34 mAb (Becton Dickinson)
and fluorescein isothiocyanate-conjugated mAbs for humanlineage-specific Ags, including CD19 (eBioscience, San
Diego, CA, USA) and CD33 (Coulter) for the detection ofhuman stem/progenitor, lymphoid, and myeloid hematopoietic
cells, respectively.

NOD/SCID and NOG miceFemale 5-week-old NOD/Shi-scid/scid (NOD/SCID) mice werepurchased from Clea Japan (Tokyo, Japan). Female 6-week-old
NOD/Shi-scid/IL-2Rgnull(NOG) mice12 were also purchasedcfrom the Central Institute of Experimental Animals (Kawasaki,
Japan). All mice were handled under sterile conditions and weremaintained in germ-free isolators located in the Central
Laboratory Animal Facilities of Kansai Medical University. The
animal experiments were approved by the Animal Care
Committees of Kansai Medical University.

SRC assay and serial analysis of human cell engraftment
in NOD/SCID or NOG mice by flow cytometry
1–5

An SRC assay was performed using an earlier reported methodwith slight modifications.7–9,13 In this study, purified 2.5  103
to 2  104 CB-derived LinCD34 þ CD38 þ , 20–4  102 CBderived LinCD34CD38 cells, or 2–7  104 CB-derived
LinCD34 cells were transplanted by IBMI7–9 into sublethally
irradiated (250 cGy using a 137Cs-g irradiator) 8- to 10-week-old
NOD/SCID or NOG mice. The repopulation of humanhematopoietic cells in murine BMs was serially analyzed by
the aspiration method. Two to 18 weeks after transplantation,
the BMs were separately aspirated from the injected left tibia
and the contra-lateral site (right tibia) of each mouse using aheparinized tuberculin syringe and were flushed into a-medium.
In LT human hematopoietic cell reconstitution experiments
using NOG mice, BMs were serially aspirated from the contralateral sites (right tibia) of IBMI of each mouse. In some of theseexperiments, the mice were killed 5, 8, 12, and 24 weeks after
transplantation and the BMs from the pairs of femurs and tibiae
of each mouse were flushed into a-medium. The repopulation ofhuman hematopoietic cells in murine BMs was determined by

Limiting dilution analysis
To assess the frequency of SRCs in the CB-derived
LinCD34 þ CD38 þ , LinCD34 þ CD38, and LinCD34
cells, various numbers of LinCD34 þ CD38 þ cells (2500,
5000, 10 000, and 15 000; n ¼ 32), LinCD34 þ CD38 cells(20, 50 and 100; n ¼ 39), and LinCD34 cells (5000, 20 000,
and 40 000; n ¼ 21) were transplanted to NOD/SCID mice by
IBMI as reported earlier.7,8

Statistical analysis
In the limiting dilution analysis, the frequencies of SRCs were
calculated using the Poisson statistics as reported earlier.3,5 For
each cell population, the data from several limiting dilution
experiments were pooled and analyzed by applying the Poisson
statistics to the single-hit model.14 The frequency of SRC in eachcell population was calculated using the weighted mean (WM)
estimator, as reported.15
In the analysis of the in vivo migration pattern, the difference
between the ratios of the human cell repopulation at the site of
injection and the contra-lateral site of injection was checked by
Fisher’s exact test.

Results

Isolation of LinCD34 þ CD38 þ , LinCD34 þ CD38,and LinCD34 cells by FACS
First, the R1 gate was set on the blast–lymphocyte window
as shown in Figure 1a. Next, LinCD45 þ CD34 þ and
LinCD45 þ CD34 cells were gated as R2 and R3 as shownin Figure 1b. These LinCD45 þ CD34 þ cells were further
LeukemiaIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al164
subdivided into two distinct populations gated as R4 and R5
based on their surface CD38 expression (Figure 1c). These threefractions were then sorted for the subsequent SRC assays.
The contamination of the LinCD34 cell fraction with
LinCD34 þ cells was ruled out by a semiquantitative reverse
transcriptase polymerase chain reaction analysis as shown in the
Supplementary Figure 1.

Limiting dilution analysis of LinCD34 þ CD38 þ ,LinCD34 þ CD38, and LinCD34 cells
Next, a limiting dilution analysis was performed to clarify thefrequency of SRCs in CB-derived LinCD34 þ CD38 þ and
LinCD34 þ CD38 cells. As shown in Figure 2a and b, limiting
dilution analyses clearly showed that the frequencies of SRCs in
CB-derived LinCD34 þ CD38 þ and LinCD34 þ CD38 cells
using IBMI were estimated to be 1/6640 and 1/44, respectively.
The frequency of SRCs in LinCD34 þ CD38 cells by tail vein
injection is reported to be approximately 1/600.3,16 On the other
hand, the frequency of SRCs in the same cell population by IBMIis also reported to be 1/44 or 1/121.16,17 Therefore, these data
are comparable with these earlier reports. According to theseresults, the IBMI method seemed to be very sensitive and could
detect the CD34 þ SRCs with 46–15-fold higher frequency thanthat by the conventional tail vein injection method. In contrast,
the frequency of CD34 SRCs in LinCD34 cells wasestimated to be 1 out of 25 523 (Figure 3c), which wasconsistent with the findings of earlier reports.7,8Characteristics of in vivo migration patterns of
CD34 þ CD38 þ , CD34 þ CD38, or CD34 SRCs
On the basis of the data obtained from these limiting dilution
analyses (Figure 2), 100 LinCD34 þ CD38 cells, 15 000–
20 000 LinCD34 þ CD38 þ cells, or 60 000–70 000 LinCD34
cells (each containing approximately 2–3 SRCs) were transplanted into primary recipient mice. Thereafter, the humanCD45 þ cell repopulation in the site of injection and the otherbones were serially investigated, separately for 2–8 weeks(Figure 3). Interestingly, CD34 þ CD38 þ SRCs completed theirmigration from the site of injection to the other bones 3 weeks
after the transplantation (Figure 3a). On the other hand,
CD34 þ CD38 SRCs began to migrate 3 weeks after thetransplantation, as shown in Figure 3b and the Supplementary
Table. At this time point, the human CD45 þ cells were detectedin the other bones of two out of four mice that received
CD34 þ CD38 SRCs. In addition, most of them completed theirmigration from the site of injection to the other bones 5 weeks
after the transplantation (Figure 3b). The median rates of humanCD45 þ cells at 5 weeks at both sites were comparable (3.5 vs2.8%) (Supplementary Table).

Figure 2 Comparison of the frequency of SRC in LinCD34 þ CD38 þ , LinCD34 þ CD38–, and LinCD34 cells. Various numbers of
LinCD34 þ CD38 þ cells (n ¼ 32; a), LinCD34 þ CD38 cells (n ¼ 39; b), and LinCD34 cells (n ¼ 21; c) were transplanted to NOD/SCID mice.
The human CD45 þ cell repopulation in the mouse BMs was analyzed by flow cytometry 8 weeks after transplantation. The frequencies of SRCs inthese three populations were as follows: 1 per 6640 LinCD34 þ CD38 þ cells (a), 1 per 44 LinCD34 þ CD38 cells (b), and 1 per 25 523
LinCD34 cells (c). For each frequency determination, the middle solid line represents the estimate fWM. The lower and the upper dotted lines
represent the 95% confidence limit of fWM.
LeukemiaIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al

165

Figure 3 In vivo migration patterns of CD34 þ CD38 þ , CD34 þ CD38–, and CD34 SRCs. On the basis of the limiting dilution analyses (Figure 2),
100 LinCD34 þ CD38 cells, 15 000–20 000 LinCD34 þ CD38 þ cells, or 60 000–70 000 LinCD34 cells (each containing approximately 2–3
SRCs) were injected into the left tibia of each mouse. The human CD45 þ cell rates in the left tibia (open circle) as well as in the other bones(closed diamond) were separately and serially analyzed. Three to seven mice were analyzed at each time point. Interestingly, CD34 þ CD38 þ andCD34 þ CD38 SRCs completed or began to migrate 3 weeks after transplantation (a, b). In contrast, CD34 SRCs first began to migrate 5 weeks
after transplantation (c). The horizontal bars show the median values. Precise data are presented in the Supplementary Table.

Figure 4 Delayed in vivo migration pattern of CD34 SRCs in comparison to that of CD34 þ CD38 SRCs. In these representative mice, 200 (fiveSRCs) of LinCD34 þ CD38 and 7  104 (three SRCs) of LinCD34 cells were injected into the left tibia of each mouse, respectively. The numberof SRCs transplanted were estimated based on the limiting dilution analyses data presented in Figure 2. The human CD45 þ cell repopulation of theinjected left tibia and other bones of these mice were analyzed 5 weeks after transplantation. (a) The mice receiving transplants of five
CD34 þ CD38 SRCs showed human cell repopulation in the injected left tibia as well as in the other bones. Both the human CD45 þ CD34 þ andCD45 þ CD34 cells are detected. (b) The mice receiving transplants of three CD34 SRCs showed human cell repopulation in the injected left
tibia; however, only CD45 þ CD34 cells were detected. In contrast, the human CD45 þ cells were not detected in the other bones.

In contrast, CD34 SRCs stayed at the site of injection until 4
weeks after the transplantation and first began to migrate toother bones 5 weeks after the transplantation. As shown in
Figure 3c and Supplementary Table, two out of five mice
showed the human cell repopulation only in the site of injection
5 weeks after transplantation. The median rates of humanCD45 þ cells at the contra-lateral site (0.3%) seemed to be lower
than that (2.4%) at the site of injection (Supplementary Table).
However, the differences between the ratios of human cell
repopulation at the contra-lateral site of injection in the micereceiving CD34 þ CD38 SRCs and CD34 SRCs were 2/4 vs 0/4
at 3 weeks and 5/6 vs 3/5 at 5 weeks, respectively. These
differences were not statistically significant (P40.05). Therefore,
further studies will be needed to elucidate the in vivo migration
patterns of these two classes of SRCs more precisely.

Next, a precise comparative analysis of the in vivo migration
patterns of CD34 þ CD38 and CD34 SRCs was performed. In
these experiments, 200 LinCD34 þ CD38 cells (containing
approximately five SRCs) and 7  104 LinCD34 cells (containing approximately three SRCs) were transplanted into eightmice. All of the three mice that received five CD34 þ CD38
SRCs showed the human CD45 þ cell repopulation in both sites.
On the other hand, three out of five mice that received three
CD34 SRCs showed the human CD45 þ cell repopulation onlyin the site of injection. As shown in Figure 4a, a representative
mouse that received transplants of five CD34 þ CD38 SRCs
showed the human CD45 þ cell repopulation in the injected left
tibiae as well as in the other bones 5 weeks after thetransplantation. In addition, human CD45 þ CD34 þ andCD45 þ CD34 cells were detected at both sites. In contrast, a
LeukemiaIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al

166

representative mouse that received three CD34 SRCs showed
the human CD45 þ cell repopulation only in the injected left
tibiae 5 weeks after the transplantation (Figure 4b, left panel). In
addition, human CD45 þ CD34 cells were only detected in theinjected left tibia. Figure 4b (right panel) clearly showed that no
human CD45 þ cells were detected in the other bones. These
results suggest that CD34 SRCs may, therefore, remain or
slowly proliferate as a CD34 phenotype at the site of injection
for at least 5 weeks.
All these findings indicated that CD34 SRC as well as
CD34 þ CD38 þ and CD34 þ CD38 SRCs could actively
migrate from the site of injection to the other bones. However,
the timing of the initiation of migration may differ between the
CD34 þ and CD34 SRCs. These results suggested, for the firsttime, that CD34 SRCs show different redistribution kinetics andalso suggest that the identified CD34 SRCs7–10 are a distinct
class of primitive HSCs in comparison to CD34 þ CD38 þ andCD34 þ CD38 SRCs.3,11,16

LT repopulation patterns of CD34 þ CD38 þ ,CD34 þ CD38, and CD34 SRCs in NOG mice

The next approach to characterize the CD34 SRCs with respect
to self-renewal potential as well as LT repopulating potential
was to serially analyze the kinetics of engraftment for 24 weeks
in the contra-lateral sites of IBMI in NOG mice that received
transplants of only one CD34 SRC. This was compared withthe repopulating pattern in mice that received 2–3
CD34 þ CD38 þ SRCs and 10 CD34 þ CD38 SRCs using NOG
mice as shown in Figure 5. The numbers of SRCs transplanted
were estimated based on the data obtained from the limiting
dilution analyses presented in Figure 2.
In these experiments, both the mice that received transplantsof CD34 þ CD38 þ and CD34 þ CD38 SRCs showed signof human cell repopulation at 5 weeks after the transplantation (Figure 5). Moreover, the mice that received
transplants of CD34 þ CD38 þ SRCs showed the peak level ofrepopulation at 5 weeks, as their repopulation rates were
continuously decreased to under the 1% level at 24 weeks after

Figure 5 LT human hematopoietic cell reconstitution in NOG mice.
A total of 2 to 3 CD34 þ CD38 þ SRCs (open triangle), 10
CD34 þ CD38 SRCs (open circle), and only 1 CD34 SRCs (closed
diamond) were injected into the left tibia of NOG mice, respectively.
The number of SRCs transplanted was estimated based on the limiting
dilution analyses data presented in Figure 2. The human CD45 þ cellrates in the contra-lateral sites of IBMI of recipient NOG mice wereserially analyzed by the aspiration method 5, 12, 18, and 24 weeks
after transplantation by flow cytometry. Data represent the mean±s.d.
of the results from five mice at each time point.
Leukemiatransplantation. However, the repopulation rates of the micethat received transplants of CD34 þ CD38 SRCs increased
during 5–12 weeks and showed the peak levels at 12 weeks after
transplantation. These repopulation levels were maintained
thereafter.
Interestingly, the mice that received transplants of CD34
SRCs did not show any sign of human cell repopulation at 5
weeks after transplantation in the contra-lateral site of IBMI as
shown in Figure 4b. The difference between the ratios of humancell repopulation at the contra-lateral site of injection in the
NOG mice receiving CD34 þ CD38 SRCs vs CD34 SRCs were
5/5 vs 0/5 at 5 weeks after the transplantation, as shown in
Figure 5, and this difference was statistically significant(P ¼ 0.00794). These findings also suggest that the timing of
the initiation of migration may differ between the CD34 þ andCD34 SRCs. However, the repopulation rates of CD34 SRCs
gradually increased at 12 weeks after the transplantation. They
showed a peak level of repopulation at 24 weeks after
transplantation. Next, the lympho-myeloid repopulating patterns were analyzed in the mice that received these three classesof SRCs. As shown in Supplementary Figure 2, both CD34
SRCs and CD34 þ CD38 SRCs showed lympho-myeloid repopulation; however, the majority of CD45 þ human cells were
CD19 þ B-lymphoid cells. In contrast, CD34 þ CD38 þ SRCs
showed only CD33 þ myeloid cell repopulation.
These results indicated that CD34 SRC, as well as
CD34 þ CD38 SRCs, could, therefore, sustain LT human cell
repopulation in NOG mice. However, the CD34 þ CD38 þ SRCs
could only support short-term (ST) human cell repopulation asreported earlier.11

Discussion
The hierarchical organization of human primitive HSC has been
extensively investigated by means of the xenotransplantation
system using NOD/SCID mice, which is a commonly usedsurrogate assay and allows for the evaluation of primitive human
lympho-myeloid reconstituting HSC in vivo.1–11,16 A number ofclonal tracking studies using retro- or lenti-viral vector haveshown that CB-derived CD34 þ cells are a heterogeneous
population and contain ST- and LT-repopulating SRCs.16,18–21
Guenechea et al.18 revealed that the type of repopulation in
NOD/SCID mice that receive transplants of retrovirally transduced CB-derived HSCs is generally oligoclonal with anextensive variability in the life span and proliferative capacityof individual SRCs. In addition, McKenzie et al.21 clearly
showed that the functionally equivalent SRC daughter cell pairs
can have distinct and unpredictable repopulation kinetics. Veryrecently, Yahata et al.22 reported that human CB-derived LTSRCs exist in a rare population of CD34 þ CD38 cells that
localized to the stem cell niche and maintained their stem cell
activities while being in a quiescent state. Moreover, clonallydistinct LT-SRCs control hematopoietic homeostasis and create
a stem cell pool hierarchy by asymmetric self-renewal divisionthat produced lineage-restricted ST-SRCs and long-lasting
LT-SRCs.22 They also showed that the CD34 þ CD38 þ ST-SRCs
are subdivided to myeloid-restricted ST-SRCs and lymphomyeloid ST-SRCs.22 All these studies clearly show the existenceof different classes of HSCs with variable proliferation and selfrenewal potentials.16,18–22
This study investigated the characteristics of CBderived primitive HSCs/HPCs, including CD34 þ CD38 þ andCD34 þ CD38 SRCs and the identified CD34 SRCs,7–10 using
the sensitive IBMI method7–9,16,17,21 to more precisely elucidateIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al

the hierarchical organization of these three classes of humanprimitive HSCs/HPCs. The IBMI method can improve thehoming efficiency;7–9,16,17 therefore, the frequency of these
SRCs in target populations increased in comparison with thoseanalyzed by the tail vein injection method.7,8,16 As reported
earlier,7,8 limiting dilution analyses showed that the frequency
of CD34 þ and CD34 SRCs in CB-derived LinCD34high andLinCD34 cells were estimated to be 1/1010 and 1/24 100,
respectively.7,8 The frequencies of SRCs in CB-derived
LinCD34 þ CD38 þ , LinCD34 þ CD38, and LinCD34 cells
were approximately 1/6600, 1/40, and 1/25 500 cells, respectively, using the IBMI (Figure 2). On the basis of these data, theestimated but comparable numbers of these three classes of
SRCs were transplanted, and their HSC characteristics werecompared using a serial analysis of human cell repopulation in
BMs of recipient mice at the site of injection as well as at thecontra-lateral sites of IBMI separately. The data clearly showedthat CD34 þ CD38 þ SRCs completed the migration from the siteof injection to the other bones 3 weeks after the transplantation,
which is at an earlier time point than that of CD34 þ CD38
SRCs (Figure 3; Supplementary Table). Interestingly, a substantial number of CD34 SRCs were retained at the site of injection
at least for 5 weeks (Figures 3 and 4) and began to migrate fromthe site of injection to the other bones thereafter. The specifichoming of circulating HSCs into the BM niche has been reported
to depend on their responsiveness to stromal-derived factor-1.23
In addition, the stromal-derived factor-1 and its receptor CXCR4
were found to have a critical function in murine BM engraftment
by human SRCs.24 An earlier study7 showed that CB-derived
CD34 SRCs showed poor stromal-derived factor-1/CXCR4mediated homing abilities, possibly because of their low levelsof homing receptor expression, including CXCR4, CD62L, andCD106. Therefore, the different expression patterns of thesehoming receptors on CD34 þ and CD34 SRCs may affect thein vivo redistribution and homing kinetics of these three classesof SRCs. Moreover, homing receptor expression of CD34 SRCs
may be changed/modified after their homing into the BM nicheusing the IBMI method. However, the molecular mechanisms
involved in this migratory (redistribution and homing) process
have yet to be clarified.
More importantly, this study clearly showed that only one(estimated number) human CB-derived CD34 SRCs could
sustain human lympho-myeloid cell repopulation for at least 24
weeks after the transplantation (Figure 5). It is well documented
that more primitive HSCs, which show a higher self-renewal
potential, can sustain the human cell repopulation in NOD/SCID or NOG mice much longer than differentiated HSCs/
HPCs.11,18,19,22 On the basis of this concept, CD34 þ CD38 aswell as CD34 SRCs could sustain up to 24 weeks of humanhematopoiesis, thus suggesting that both SRCs are primitive
HSCs. In addition, CD34 SRCs showed different redistribution
and homing kinetics in comparison to CD34 þ CD38 SRCs.
All these findings again suggest that the identified humanCB-derived CD34 SRCs represent a novel class of primitive
human HSC.7–10
Primitive LT-HSCs in mice have been reported to lack CD34
expression.25 However, Ogawa proposed the concept that theexpression of CD34 by murine HSCs is reversible.26 On theother hand, human BM-derived CD34 þ HSCs have beenreported to convert to CD34 HSCs in vivo.27,28 Both thesestudies showed that the conversion of the CD34 expression
occurs at least 8–15 months after the transplantation of purified
LinCD34 þ or LinCD34 þ CD38 cells in either preimmune
fetal sheep or homozygous bg.bg/nu.nu/xid.xid (bnx) mice.
These studies indicate the possibility that the human BM-derived

CD34 þ cell population can act as a reservoir for the generationof CD34 cells. Conversely, the human BM-derived CD34 cell
population generates CD34 þ -repopulating cells in vivo, thus
indicating that the expression of CD34 antigen can be reversible
on human BM-derived HSCs.27,28 As we reported earlier,7
human CB-derived CD34 SRCs generate CD34 þ SRCs in vitro
as well as in vivo. In contrast to BM-derived HSCs,27,28 humanCB-derived CD34 þ SRCs do not convert to CD34 SRCs for atleast 16 weeks after transplantation.7 However, it remains to be
determined whether human CB-derived CD34 þ SRCs canconvert to CD34 SRCs. Therefore, much longer observation
periods (41 year) will thus be needed, as suggested in earlier
reports.27,28
Interestingly, human BM-derived LinCD34 cells did notshow any SRC activity when they were directly transplanted into
NOD/SCID mice by IBMI (Sonoda et al. unpublished data).
These data are consistent with the findings of a recent report,29
which describes that mouse BM stromal cell line, HESS-5,
engineered to produce angiopoietin-1, supports the induction ofhematopoietic activity in human BM-derived CD34 cells, thus
suggesting that the characteristics of CD34 LT-HSC found in
human CB and BM are different classes of primitive HSC.
In summary, this study showed, for the first time, that the
CD34 SRCs show different in vivo redistribution kinetics incomparison to CD34 þ CD38 þ and CD34 þ CD38 SRCs.
In addition, the earlier study7 did not elucidate whether the
CD34 cell population obtained from primary mice transplanted with CB-derived LinCD34 cells still contain CD34
HSCs, which cannot home into the BM niche in secondary
recipients even by IBMI. Further studies are now underway to
examine this important issue. To more precisely elucidate CBderived CD34 SRCs, it is very important to identify a specificpositive marker for the higher degree purification of this rareclass of primitive HSCs. Additional studies are also currently
being conducted to clarify the candidate positive markers forthese human primitive HSCs.

167

Conflict of interest
The authors declare no conflict of interest.

Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research
C (grant Nos. 19591144 and 21591251) from the Ministry of
Education, Science and Culture of Japan, a grant from Haiteku
Research Center of the Ministry of Education, a grant from the
Science Frontier Program of the Ministry of Education, a grantfrom the 21st Century Center of Excellence (COE) program of the
Ministry of Education, a grant from the Promotion and Mutual Aid
Corporation for Private Schools of Japan, a grant from Kansai
Medical University (Research grant B), a grant from the Japan
Leukemia Research Foundation, a grant from the Mitsubishi
Pharma Research Foundation, and a grant from the Takeda
Science Foundation.

References
1 Dick JE. Normal and luekemic human stem cells assayed in SCID
mice. Semin Immunol 1996; 8: 197–206.
2 Larochelle A, Vormoor J, Hanenberg H, Wang JCY, Bhatia M,
Lapidot T et al. Identification of primitive human hematopoietic
cells capable of repopulating NOD/SCID mouse bone marrow:
implications for gene therapy. Nat Med 1996; 2: 1329–1337.
LeukemiaIn vivo dynamics of CD34 SCID-repopulating cell
T Kimura et al168
3 Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE. Purification ofprimitive human hematopoietic cells capable of repopulating
immune-defficient mice. Proc Natl Acad Sci USA 1997; 94:
5320–5325.
4 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.
5 Wang JCY, Doedens M, Dick JE. Primitive human hematopoietic
cells are enriched in cord blood compared with adult bone marrowor mobilized peripheral blood as measured by quantitative in vivo
SCID-repopulating cell assay. Blood 1997; 89: 3919–3924.
6 Kim DK, Fujiki Y, Fukushima T, Ema H, Shibuya A, Nakauchi H.
Comparison of hematopoietic activities of human bone marrow
and umbilical cord blood CD34 positive and negative cells. StemCells 1999; 17: 286–294.
7 Wang J, Kimura T, Asada R, Harada S, Yokota S, Kawamoto Y et al.
SCID-repopulating cell activity of human cord blood-derived
CD34- cells assured by intra-bone marrow injection. Blood
2003; 101: 2924–2931.
8 Kimura T, Wang J, Matsui K, Imai S, Yokoyama S, Nishikawa M
et al. Proliferative and migratory potentials of human cord bloodderived CD34 severe combined immunodeficiency repopulating
cells that retain secondary reconstituting capacity. Int J Hematol
2004; 79: 328–333.
9 Kimura T, Asada R, Wang J, Kimura T, Morioka M, Matsui K et al.
Identification of long-term repopulating potential of human cord
blood-derived CD34flt3 severe combined immunodeficiencyrepopulating cells by intra-bone marrow injection. Stem Cells
2007; 25: 1348–1355.
10 Sonoda Y. Immunophenotype and functional characteristics ofhuman primitive CD34-negative hematopoietic stem cells: thesignificance of the intra-bone marrow injection. J Autoimmunity
2008; 30: 136–144.
11 Hogan CJ, Shpall EJ, Keller G. Differential long-term and multilineage engraftment potential from subfractions of human CD34+
cord blood cells transplanted into NOD/SCID mice. Proc Natl
Acad Sci USA 2002; 99: 413–418.
12 Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K
et al. NOD/SCID/gcnull mouse: an excellent recipientmouse model for engraftment of human cells. Blood 2002; 100:
3175–3182.
13 Kimura T, Minamiguchi H, Wang J, Kaneko H, Nakagawa H,
Fujii H et al. Impaired stem cell function of CD34+ cells selectedby two different immunomagnetic beads systems. Leukemia 2004;
18: 566–574.
14 Porter EH, Berry RJ. The efficient design of transplantable tumor
assays. Br J Cancer 1964; 17: 583–595.
15 Taswell C. Limiting dilution assays for the determination of
immunocompetent cell frequencies. I. Data analysis. J Immunol
1981; 126: 1614–1619.

16 Yahata T, Ando K, Sato T, Miyatake H, Nakamura Y, Muguruma Y
et al. A highly sensitive strategy for scid-repopulating cell assay bydirect injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow. Blood 2003; 101: 2905–2913.
17 McKenzie JL, Takenaka K, Gan OI, Doedens M, Dick JE. Low
rhodamine 123 retention identifies long-term human hematopoietic stem cells within the Lin-CD34+CD38 population. Blood
2007; 109: 543–545.
18 Guenechea G, Gan OI, Dorrell C, Dick JE. Distinct classes ofhuman stem cells that differ in proliferative and self-renewal
potential. Nat Immunol 2001; 2: 75–82.
19 Mazurier F, Gan OI, McKenzie JL, Doedens M, Dick JE.
Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment
and culture-induced stem cell impairment. Blood 2004; 103:545–552.
20 Yahata T, Yumino S, Seng Y, Miyatake H, Uno T, Muguruma Y
et al. Clonal analysis of thymus-repopulating cells presents direct
evidence for self-renewal division of human hematopoietic cells.
Blood 2006; 108: 2446–2454.
21 McKenzie JL, Gan OI, Doedens M, Wang JCY, Dick JE. Individual
stem cells with highly variable proliferation and self-renewal
properties comprise the human hematopoietic stem cell compartment. Nat Immunol 2006; 7: 1225–1233.
22 Yahata T, Muguruma Y, Yumino S, Sheng Y, Uno T, Matsuzawa H
et al. Quiescent human hematopoietic stem cells in the bonemarrow niches organize the hierarchical structure of hemtopoiesis.
Stem Cells 2008; 26: 3228–3236.
23 Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL.
Hematopoietic stem cells are uniquely selective in their migratory
response to chemokines. J Exp Med 2002; 195: 1145–1154.
24 Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T et al.
Dependence of human stem cell engraftment and repopulation of
NOD/SCID mice on CXCR4. Science 1999; 283: 845–848.
25 Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term
lymphohematopoietic reconstitution by a single CD34-low/
negative hematopoietic cell. Science 1996; 273: 242–245.
26 Ogawa M. Changing phenotypes of hematopoietic stem cells. Exp
Hematol 2002; 30: 3–6.
27 Zanjani ED, Almeida-Porada G, Livingston AG, Zeng H-Q, Ogawa
M. Reversible expression of CD34 by adult human bone marrow
long-term engrafting hematopoietic stem cells. Exp Hematol 2003;
31: 406–412.
28 Dao MA, Arevalo J, Nolta JA. Reversibility of CD34 expression onhuman hematopoietic stem cells that retain the capacity forsecondary reconstitution. Blood 2003; 101: 112–118.
29 Nakamura Y, Yahata T, Muguruma Y, Uno T, Sato T, Matsuzawa H
et al. Angiopoietin-1 supports induction of hematopoietic activity
in human CD34 bone marrow cells. Exp Hematol 2007; 35:
1872–1883.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia