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Identification of Long-Term Repopulating Potential of Human Cord
Blood-Derived CD34 –flt3– Severe Combined Immunodeficiency-Repopulating
Cells by Intra-Bone Marrow Injection
Takafumi Kimura, Rumiko Asada, Jianfeng Wang, Takashi Kimura, Miho Morioka,
Kazuo Matsui, Katsuya Kobayashi, Kae Henmi, Shiro Imai, Masakazu Kita, Takashi
Tsuji, Yutaka Sasaki, Susumu Ikehara and Yoshiaki Sonoda
Stem Cells 2007;25;1348-1355; originally published online Feb 15, 2007;
DOI: 10.1634/stemcells.2006-0727

TISSUE-SPECIFIC STEM CELLS
Identification of Long-Term Repopulating Potential of Human Cord
Blood-Derived CD34ⴚflt3ⴚ Severe Combined ImmunodeficiencyRepopulating Cells by Intra-Bone Marrow Injection
TAKAFUMI KIMURA,a RUMIKO ASADA,a JIANFENG WANG,a TAKASHI KIMURA,a,g MIHO MORIOKA,aKAZUO MATSUI,b KATSUYA KOBAYASHI,c KAE HENMI,c SHIRO IMAI,c MASAKAZU KITA,d TAKASHI TSUJI,eYUTAKA SASAKI,a SUSUMU IKEHARA,f YOSHIAKI SONODAaa

Key Words. Flt3 • Severe combined immunodeficiency-repopulating cell • Intra-bone marrow injection • Cord blood • Hematopoiesis

ABSTRACT
Recently, we have identified human cord blood (CB)derived CD34-negative (CD34ⴚ) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using theintra-bone marrow injection (IBMI) method (Blood 2003;
101:2924). In contrast to murine CD34ⴚ KitⴙSca-1ⴙLineageⴚ (KSL) cells, human CB-derived LinⴚCD34ⴚ cells
did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of
flt3 in our identified human CB-derived CD34ⴚ SRCs.
Both CD34ⴙflt3ⴙ/ⴚ cells showed SRC activity. In the
CD34ⴚ cell fraction, only CD34ⴚflt3ⴚ cells showed distinct SRC activity by IBMI. Although CD34ⴙflt3ⴙ cells
showed a rather weak secondary repopulating activity,
CD34ⴙflt3ⴚ cells repopulated many more secondary re-cipient mice. However, CD34ⴚflt3ⴚ cells repopulated allof the secondary recipients, and the repopulating rate wasmuch higher. Next, we cocultured CD34ⴚflt3ⴚ cells withthe murine stromal cell line HESS-5. After 1 week, significant numbers of CD34ⴙflt3ⴙ/ⴚ cells were generated,
and they showed distinct SRC activity. These results indicated that CB-derived CD34ⴚflt3ⴚ cells produced
CD34ⴙflt3ⴚ as well as CD34ⴙflt3ⴙ SRCs in vitro. The
present study has demonstrated for the first time that
CB-derived CD34ⴚ SRCs, like murine CD34ⴚ KSL cells,
do not express flt3. On the basis of these data, we proposethat the immunophenotype of very primitive long-term
repopulating human hematopoietic stem cells is
LinⴚCD34ⴚc-kitⴚflt3ⴚ. STEM CELLS 2007;25:1348 –1355

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION
It is well documented that the tyrosine kinase receptors c-kit andflt3 are expressed and function in early mouse [1–10] andhuman hematopoiesis [1, 11–21]. Moreover, their respective
ligands, stem cell factor (SCF) and flt3 ligand (FL), synergistically act with each other and play an important role in theregulation (generation, maintenance, proliferation, differentiation, and expansion) of early stages of murine and humancandidate hematopoietic stem cells (HSCs) [1–21]. The mostprimitive HSCs in mammals, including mice and humans, havelong been believed to be CD34 antigen-positive (CD34⫹) [22].
However, Osawa et al. [23] revealed that murine long-term
lymphohematopoietic reconstituting HSCs are lineage markernegative (Lin⫺) c-kit⫹Sca-1⫹CD34-low/negative (CD34lo/⫺
KSL). In a murine model, it was recently reported that flt3⫺

KSL cells supported long-term multilineage hematopoietic reconstitution [24]. In contrast, flt3⫹ KSL cells are progenitors forthe common lymphoid stage [24]. These flt3⫹ KSL cells havealso been shown to lack erythro-megakaryocytic potential [25].
This notion was supported by the other reports that mice deficient in the expression of flt3 or FL showed deficient lymphopoiesis [26 –28].
Recently, using the intra-bone marrow injection (IBMI)
method, we have successfully identified human cord blood(CB)-derived CD34-negative (CD34⫺) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive
lymphoid and myeloid repopulating ability [29]. These CD34⫺
SRCs seemed to be more primitive HSCs than CD34⫹ SRCs
[29, 30]. They could home into the BM niche only by IBMI,
because they expressed lower levels of homing receptors, including CXCR4, and had poor SDF-1/CXCR4-mediated migration ability [29]. In contrast to the murine candidate HSCs

Correspondence: Yoshiaki Sonoda, M.D., Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical
Science, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan. Telephone: ⫹81-6-6993-9435; Fax: ⫹81-6-6992-3522; e-mail:sonoda@takii.kmu.ac.jp Received November 8, 2006; accepted for publication February 6, 2007; first published online in STEM CELLSEXPRESS February 15, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0727

STEM CELLS 2007;25:1348 –1355 www.StemCells.com

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Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai
Medical University, Moriguchi, Osaka, Japan; bDepartment of Gynecology and Obstetrics, Fukuda Hospital,
Kumamoto, Japan; cDepartment of Obstetrics and Gynecology, Aizenbashi Hospital, Osaka, Japan; dDepartment ofMicrobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, Japan;eDepartment of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba, Japan; fFirst
Department of Pathology, Transplantation Center, gFirst Department of Internal Medicine, Kansai Medical
University, Moriguchi, Osaka, JapanKimura, Asada, Wang et al.

MATERIALS AND METHODS
Collection of CB Samples and Processing
CB samples were obtained from normal full-term deliveries withsigned informed consent and approved by the institutional review
boards of Kansai Medical University and Kyoto Prefectural University of Medicine. CB-derived mononuclear cells (MNCs) wereisolated using Ficoll-Paque (Amersham Biosciences AB, Uppsala,Sweden; http://www.amersham.com) density gradient centrifugation. The MNCs were further enriched by negative depletion ofeight lineage-positive cells, including CD3, CD14, CD16, CD19,
CD24, CD56, CD66b, and Glycophorin A using a StemSep device(StemCell Technologies, Vancouver, BC, Canada; http://www.
stemcell.com), as reported previously[29, 30].

Purification of LinⴚCD34ⴙflt3ⴙ/ⴚ and
LinⴚCD34ⴚflt3ⴙ/ⴚ Cells
The above-mentioned lineage-negative (Lin⫺) cells were stainedwith fluorescein isothiocyanate (FITC)-conjugated anti-CD45
monoclonal antibody (mAb) (Beckman Coulter, Fullerton, CA,
http://www.beckmancoulter.com), PC5-conjugated anti-CD34 mAb(Beckman Coulter), and biotinylated anti-flt3 mAb (M22, Immunex, Seattle, http://immunex.com) followed by incubation withstreptavidin-phycoerythrin (SA-PE; Becton Dickinson, Franklin
Lakes, NJ, http://www.bd.com), as reported previously [20, 29, 30].
These stained cells were then sorted into four fractions, including
CD34⫹flt3⫹/⫺ and CD34⫺flt3⫹/⫺ cells, as shown in Figure 1C
using a FACSVantage (Becton Dickinson) as reported [29, 30]. The
viability of these sorted cells was consistently more than 99%.
Approximately 80% of the CD34⫹ cell fraction in these Lin⫺

www.StemCells.com

Figure 1. Expression of flt3 or c-kit receptor on cord blood-derived
Lin⫺ cells. (A): The forward scatter/SSC profile of immunomagnetically separated Lin⫺ cells. The R1 gate was set on the lymphocyte
window. (B): Lin⫺CD45⫹CD34⫹/⫺ cells present in R1 gate weregated as R2. (C): The expression pattern of CD34 and flt3 on R2
gated cells is shown. Cells residing in the four cell fractions wereclassified as Lin⫺CD34⫹flt3⫹, Lin⫺CD34⫹flt3⫺, Lin⫺CD34⫺flt3⫹,and Lin⫺CD34⫺flt3⫺ cells, respectively. Each sorting window isshown as a solid square. Figures in upper right corner show percentages of cells in each quadrant. (D): The expression pattern of c-kit on
Lin⫺CD34⫹/⫺ cells in a separate experiment. Abbreviations: FITC,
fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin;
SSC, side scatter.

cells expressed flt3 receptor. On the other hand, only 30% of the
CD34⫺ cell fraction in the same Lin⫺ cells expressed thisreceptor. In separate experiments, we stained these immunomagnetically separated Lin⫺ cells with 13 FITC-conjugated lineagespecific mAbs as reported previously [29], PC5-conjugated anti-CD34
mAb (Beckman Coulter), and PE-conjugated anti-c-kit mAb (Beckman
Coulter) and examined the expression pattern of c-kit receptor (Fig.
1D).

Clonal Cell Culture
Human colony-forming cells (CFCs) were assayed using our standard methylcellulose cultures as reported previously [18 –21, 29, 30,
33, 34]. Briefly, 200 or 500 sorted Lin⫺CD34⫹/⫺flt3⫹/⫺ cells wereplated in 1 ml of culture containing 1.2% methylcellulose (Shinetsu
Chemical, Tokyo, http://www.shinetsu.co.jp/e), 30% fetal calf serum (FCS; Hyclone, Laboratories, Logan, UT, http://www.hyclone.
com), 1% bovine serum albumin (Sigma-Aldrich, St Louis, http://
www.sigmaaldrich.com), 5 ⫻ 10⫺5 mol/L 2-mercaptoethanol(Sigma-Aldrich), and various recombinant human (rh) cytokines,
including SCF, interleukin (IL)-3, granulocyte macrophage colonystimulating factor (GM-CSF), granulocyte colony-stimulating factor(G-CSF), and erythropoietin (Epo) in 35-mm Lux suspension culture dishes (Nunc, Rochester, NY, http://www.nuncbrand.com). For
culture of megakaryocyte colony-forming cell (CFU-Meg), 10%
platelet-poor plasma (PPP) [33] instead of 30% FCS, and rh thrombopoietin (TPO) were used. Cytokines, including G-CSF, Epo, and
TPO were provided by Kirin Brewery Company (Takasaki, Japan,
http://www.kirin.com). SCF, IL-3, and GM-CSF were purchasedfrom R&D Systems Inc. (Minneapolis, http://www.rndsystems.
com). Dishes were incubated at 37°C in a fully-humidified atmosphere flushed with a combination of 5% CO2, 5% O2, and 90% N2.
On days 12–14 of incubation, all colonies were scored under aninverted microscope according to their typical morphologic features, as reported elsewhere [18 –21, 29, 30, 33]. CFU-Meg-derived
pure megakaryocyte colonies were identified in situ as clusters oflarge cells, which were highly refractile and showed irregular contour and hyaline nongranulated cytoplasm. The types of colonies
identified in situ were granulocyte (CFU-G), macrophage (CFUM), granulocyte/macrophage (CFU-GM), erythroid burst (BFUE), erythrocyte-containing mixed (CFU-Mix), and the abovementioned CFU-Meg. The numbers of all types of hematopoietic
colonies were determined as the mean of three independent
experiments.

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(CD34⫺ KSL cells) [23], our identified CD34⫺ SRCs did not
express detectable levels of c-kit tyrosine kinase receptor byflow cytometry. However, the degree to which flt3 is expressedon human HSCs, including CD34⫹ and CD34⫺ SRCs, which
are capable of in vivo lymphomyeloid reconstitution, has notbeen fully elucidated.
Until now, a number of studies have reported that flt3 isexpressed and functioned in the human CD34⫹ hematopoietic
progenitor cells [11–17, 19, 20], including long-term cultureinitiating cells (LTC-ICs) [15, 20, 31]. However, only tworeports have demonstrated, using the conventional intravenous
injection method, that human CB- and bone marrow (BM)derived CD34⫹ HSCs capable of multilineage reconstitution innonobese diabetic (NOD)/SCID mice express flt3 tyrosine kinase receptor [31, 32].
In this study, we have investigated, using the IBMI method,
the function of flt3, which is expressed in early mouse [1–10]
and human [11–21] hematopoiesis like c-kit, in our identified
very primitive human CB-derived CD34⫺ SRCs [29, 30], as
well as more committed CD34⫹ SRCs. Our data clearly demonstrate that part of human CB-derived CD34⫹ SRCs express
flt3, as reported previously [31, 32]. However, only
CD34⫹flt3⫺ cells showed significant secondary repopulating
ability, even when a comparable number of CD34⫹flt3⫺ cells as
CD34⫹flt3⫹ cells was transplanted. Moreover, CD34⫺flt3⫺
cells showed distinct and potent SRC activity by IBMI, and they
showed high and efficient secondary repopulating ability compared with CD34⫹flt3⫺ cells. The CD34⫺flt3⫺ cells also produced CD34⫹flt3⫺ and CD34⫹flt3⫹ SRCs after the coculture
with the murine stromal cell line HESS-5 in vitro, suggestingthat these CD34⫺flt3⫺ cells contained very primitive humanCB-derived HSCs. The results of the present study are consistent with recent studies of the significance of flt3 expression in murine primitive hematopoiesis [24 –28] and provide
a new concept of hierarchy in the human primitive HSC
compartment.

1349

1350

IBMI of Purified Cells
IBMI was carried out as reported previously [29, 30, 35]. 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 tothe anterior. A 27-gauge needle was inserted into the joint surfaceof the tibia through the patellar tendon and then inserted into the
BM cavity. Using a Hamilton’s microsyringe, the number-specified
donor cells per under 10 ␮l of ␣-medium were carefully and slowly
injected from the bone hole into the BM cavity.

SCID-Repopulating Cell Assay

Analysis of Human Cell Engraftment in NOD/SCID
Mice by 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) byflow cytometry. The cells were also stained with PE-conjugatedanti-human CD34 mAb (Becton Dickinson), and FITC-conjugatedmAbs for human lineage-specific Ags, including CD19 (eBioscience, San Diego, http://www.ebioscience.com), and CD33
(Beckman Coulter) for the detection of human lymphoid and myeloid hematopoietic cells, respectively.

Secondary Transplantation
For secondary transplantations, murine BM cells were obtained
from the pairs of femurs, tibiae, and humeri of moderately engrafted
primary recipient mice 8 –12 weeks after transplantation with 3 ⫻103 to 5 ⫻ 103 Lin⫺CD34⫹flt3⫹, 4 ⫻ 103 to 5 ⫻ 103
Lin⫺CD34⫹flt3⫺, or 2 ⫻ 104 to 3 ⫻ 104 Lin⫺CD34⫺flt3⫺ cells,
respectively. The human cell repopulation rates in the primary
recipients’ BMs were comparable and approximately 4%– 8%.
Whole BM cells were transplanted by IBMI into sublethally (250cGy) irradiated secondary recipient mice. Eight to 10 weeks after
transplantation, the presence of human CD45⫹ cells in the secondary recipients’ BMs was analyzed by flow cytometry, as describedfor primary transplantation.

Coculture with HESS-5 Cells and SRC Activity of
Culture-Generated CD34ⴙflt3ⴙ/ⴚ Cells
A total of 5 ⫻ 104 purified Lin⫺CD34⫺flt3⫺ cells per 12.5-cm2
culture flask (BD Falcon; Becton Dickinson) onto preestablishedirradiated HESS-5 [39] layers in StemPro-34 medium (Gibco Laboratories, Grand Island, NY, http://www.invitrogen.com) and a
cocktail of cytokines, including 300 ng/ml SCF (R&D), 300 ng/ml
TPO (Kirin), 10 ng/ml IL-3 (R&D), 10 units/ml IL-6 (provided by

Dr. Akira Okano, Ajinomoto Co. Inc., Yokohama, Japan, http://
www.ajinomoto.com), 10 ng/ml G-CSF (Kirin), and 5% FCS (Hyclone). After 1 week, all cells were collected by vigorous pipetting,and stained with PC5-conjugated anti-CD34 mAb (Beckman
Coulter) and biotinylated anti-flt3 mAb (Immunex) as mentioned
herein. Cells were then stained with SA-PE (Becton Dickinson).
The rates of CD34⫹flt3⫹/⫺ cells were analyzed by flow cytometry.
Simultaneously, these CD34⫹flt3⫹/⫺ cells were separately obtainedby cell sorting (FACSVantage) for the detection of SRC activity.
One to 2 ⫻ 104 CD34⫹flt3⫹ or 2 ⫻ 104 to 4 ⫻ 104 CD34⫹flt3⫺
cells were transplanted by IBMI into sublethally (250 cGy) irradiated recipient mice. Eight weeks after transplantation, the presenceof human CD45⫹ cells in the recipients’ BMs was analyzed by flow
cytometry, as described for primary transplantation.

Statistical Analysis
The significance of differences in the SRC assays and the numbersof hematopoietic colonies was determined using the Mann-Whitney
U test and the two-tailed Student’s t test, respectively.

RESULTS
Expression of flt3 and c-kit Receptors on
LinⴚCD45ⴙCD34ⴙ/ⴚ Cells
First, we depleted the eight lineage-positive cells from CBderived MNCs using the immunomagnetic beads system [29,30]. Then, Lin⫺CD45⫹CD34⫹/⫺ cells were gated as R2 asshown in Figure 1B. These Lin⫺CD45⫹ cells were subdivided
into four distinct populations on the basis of their surface CD34
and flt3 expression (Fig. 1C). We sorted these four fractions
for further stem cell characterization. The phenotypic purityof the sorted cells consistently exceeded 98% when checkedusing postsorting flow cytometric analysis (data not shown).
Importantly, these Lin⫺CD34⫺ cells did not express detectable levels of c-kit receptors by flow cytometry, as shown in
Figure 1D.

Characteristics of Colony-Forming Capacity by CBDerived LinⴚCD34ⴙ/ⴚflt3ⴙ/ⴚ Cells
The colony-forming capacities of these four fractions were quite
different. The plating efficiency of each Lin⫺CD34⫹flt3⫹ or
Lin⫺CD34⫹flt3⫺ cell fraction was approximately 50% andcomparable (Fig. 2A). Lin⫺CD34⫹flt3⫹ cells contained approximately 81% CFU-GM, 17% BFU-E, and 2% CFU-Mix. In
contrast, Lin⫺CD34⫹flt3⫺ cell fraction contained 21% CFUGM,
66%
BFU-E,
and12%
CFU-Mix.
The
Lin⫺CD34⫺flt3⫹/⫺ cell fractions showed almost no colony
formation (data not shown). On the other hand, the vast
majority of CFU-Megs (more than 90%) were detected in the
Lin⫺CD34⫹flt3⫺ cell fraction (Fig. 2B).
These results clearly demonstrate that CB-derived
Lin⫺CD34⫹flt3⫹ cells display weak erythroid and megakaryocytic potentials. These findings were consistent with a recent
study in which murine flt3⫹ KSL cells failed to produce significant erythroid and megakaryocytic progeny [25].

SRC Activity and Lymphomyeloid-Reconstituting
Capacity of CB-Derived LinⴚCD34ⴙ/ⴚflt3ⴙ/ⴚ Cells
by IBMIIn this study, we have investigated the function of flt3 in ouridentified human CB-derived CD34⫺ SRCs. First, we studied
the SRC activity of CB-derived Lin⫺CD34⫹flt3⫹/⫺ or
CD34⫺flt3⫹/⫺ cells using IBMI, as shown in Figure 3. Both
CD34⫹flt3⫹/⫺ cells repopulated all 20 recipient mice (10 miceeach). The level of human CD45⫹ cells in the murine BMs that

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An SRC assay was performed using the methods reported previously [36, 37] with modifications [29, 30, 38]. Five-week-old NOD/
Shi-scid/scid (NOD/SCID) mice were purchased from Clea Japan(Tokyo, Japan, http://www.clea-japan.com). The animal experiments were approved by the Animal Care Committees of Kansai
Medical University and Kyoto Prefectural University of Medicine.
All mice were handled in sterile conditions and maintained in
germ-free isolators located in the Central Laboratory Animal Facilities of Kansai Medical University and Kyoto Prefectural Universityof Medicine. In this study, purified 3 ⫻ 104 to 5 ⫻ 104 CB-derived
Lin⫺CD34⫹flt3⫹/⫺, or 2 ⫻ 104 to 7 ⫻ 104 CB-derived
Lin⫺CD34⫺flt3⫹/⫺ cells were transplanted by IBMI into sublethally irradiated (250 cGy using a 137Cs-␥ irradiator) 8 –12-weekold mice. As we reported previously [29, 30], CB-derived CD34⫺
SRCs were detected only by the IBMI technique. Moreover, therepopulation rate of CD34⫹ SRCs by IBMI was significantly higher
than that by the conventional tail-vein injection method [29]. Therefore, we used the IBMI technique to analyze SRC activities of
Lin⫺CD34⫹/⫺Flt3⫹/⫺ cells in this study. The mice were killed
8 –12 weeks after transplantation, and the BMs from the pairs of
femurs, tibiae, and humeri of each mouse were flushed into ␣-medium. The rates of human CD45⫹ cells in the murine BMs wereanalyzed by flow cytometry (FACS Calibur; Becton Dickinson) as
described in the next section. Mice were scored as positive if over
0.1% of total murine BM cells were human CD45⫹.

SRC Activity of CD34⫺Flt3⫺ Cells by IBMI

Kimura, Asada, Wang et al.

1351

Table 1. Lymphomyeloid reconstitution abilities ofCD34ⴙflt3ⴙ/ⴚ and CD34ⴚflt3ⴚ severe combined
immunodeficiency repopulating cells by intra-bone marrow(BM) injection
%
Cells
⫹

⫹

CD34 flt3

CD34⫹flt3-

Figure 3. Severe combined immunodeficiency-repopulating cell activities of Lin⫺CD34⫹/⫺flt3⫹/⫺ cells by intra-BM injection (IBMI). (A):Each mouse transplanted with designated numbers of cord bloodderived Lin⫺CD34⫹flt3⫹, Lin⫺CD34⫹flt3⫺, Lin⫺CD34⫺flt3⫹, and
Lin⫺CD34⫺flt3⫺ cells was sacrificed 8 –12 weeks after transplantation.
Closed circles represent the repopulation rates in total murine BMs by
the IBMI, respectively. Horizontal bars represent each median of therepopulation rates. (B–D): The human CD45⫹ cell reconstitution in
the representative mouse presented in (A) received transplants of
CD34⫹flt3⫹ (B), CD34⫹flt3⫺ (C), and CD34⫺flt3⫺ (D) cells, respectively. Percentages of cells in each quadrant are presented in the upper
left corner. Abbreviations: BM, bone marrow; PE, phycoerythrin.

received transplants of CD34⫹flt3⫹ cells (n ⫽ 10; 29.3% to90.8%; median, 65.4%) was higher than those that received
transplants of CD34⫹flt3⫺ cells (n ⫽ 10; 0.3% to 45.1%;
median, 16.9%).
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CD34⫺flt3⫺

CD19

5.9
4.5
2.8
1.3
1.1
0.5
7.4
3.3
1.9

44.4
41.5
29.5
16.9
6.1
2.1
19.3
21.1
13.4

Each mouse transplanted with designated numbers of cord bloodderived Lin⫺CD34⫹flt3⫹, Lin⫺CD34⫹flt3⫺, and
Lin⫺CD34⫺flt3⫺ cells was sacrificed 8 to 12 weeks after
transplantation. First, the R1 gate was set on the total murine BM
cells obtained from these representative mice, and then humanCD45⫹ cells were gated as R2. Expression of lineage markers,
including CD19 (lymphoid) and CD33 (myeloid) on the R2 gatedcells, was analyzed by three color flow cytometry. Boldfacing
represents each median of the repopulation rates.

On the other hand, the seven mice that received transplants of
CD34⫺flt3⫹ cells did not show human cell repopulation. Only
CD34⫺flt3⫺ cells repopulated all seven recipient mice, and thelevel of human CD45⫹ cells in the murine BMs was 4.1% to 63.3%(median, 37.9%). These results indicated for the first time that the
CB-derived Lin⫺CD34⫺flt3⫺ cell population contained SRCs, asdetected by IBMI.
To further evaluate the function of flt3 expression in CD34⫹
and CD34⫺ SRCs, we studied their lymphomyeloid reconstitution abilities using IBMI. In our SRC assay system, all NOD/SCID mice transplanted either with 3 ⫻ 104 to 5 ⫻ 104
Lin⫺CD34⫹flt3⫹/⫺ cells or 5 ⫻ 104 to 7 ⫻ 104
Lin⫺CD34⫺flt3⫺ cells by IBMI showed signs of human cellengraftment. The analyses of the three representative mice transplanted either with Lin⫺CD34⫹flt3⫹/⫺ cells or Lin⫺
CD34⫺flt3⫺ cells clearly indicate that these three classes of
SRCs have an extensive differentiation capacity to B-lymphoid(CD19) and myeloid (CD33) lineages in vivo (Table 1).
Next, the percentages of lineage-positive cells expressing
CD19 and CD33 were compared (Table 1). These results demonstrated that all three classes of SRCs could supply lymphoidas well as myeloid cells at 8 –12 weeks after the transplantation.
Interestingly, CD34⫹flt3⫹ SRCs showed a lymphoid-dominant
repopulation pattern compared with the other two classes of
SRCs. These results are consistent with the notion that cells in
the Lin⫺Sca-1⫹c-kit⫹ murine HSC compartment coexpressing
flt3 tyrosine kinase receptor sustain lymphoid potential [24, 25],
and also that mice deficient in the expression of flk2/flt3 or FL
show deficient lymphopoiesis [26 –28].

Secondary Repopulating Ability of LinⴚCD34ⴙ
flt3ⴙ/ⴚ or LinⴚCD34ⴚflt3ⴚ Cells by IBMI
To further evaluate the long-term repopulating potential of thesethree populations (CD34⫹flt3⫹, CD34⫹flt3⫺, and CD34⫺flt3⫺
cells), BM cells obtained from each engrafted primary recipientmouse were assessed for their SRC activity by secondary transplantation by IBMI. Only one of six mice that received whole
BM cells obtained from primary recipient mice that received
transplants of CD34⫹flt3⫹ cells showed secondary repopulating
activity (Fig. 4). On the other hand, 83% (five of six) of the

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Figure 2. Colony-forming capacities of Lin⫺CD34⫹flt3⫹/⫺ cells. (A):The colony-forming capacities of 200 Lin⫺CD34⫹flt3⫹/⫺ cells in thepresence of stem cell factor, interleukin-3, granulocyte macrophage(GM) colony-stimulating factor (CSF), granulocyte (G) CSF, and erythropoietin. Open, shaded, closed, and gray bars represent the number of
granulocyte/macrophage colony-forming units (CFUs; including
CFU-G, CFU-macrophage, and CFU-GM), erythroid burst, CFU-Mix,
and total colony, respectively. (B): The colony-forming capacities of
500 Lin⫺CD34⫹flt3⫹/⫺ cells in the presence of thrombopoietin. Closed
bars represent the number of megakaryocyte CFUs. The numbers of all
types of colonies were determined as the mean of three independent
experiments. Vertical bars represent standard deviation, and asterisks
show statistical significance (p ⬍ .01) between the numbers of designated colonies formed by flt3⫹ and flt3⫺ cells, respectively.

CD33

SRC Activity of CD34⫺Flt3⫺ Cells by IBMI

1352

secondary recipients that received whole BM cells fromprimary recipients that received CD34⫹flt3⫺ cells could be
repopulated. Moreover, all five secondary recipient mice thatreceived whole BM cells from primary recipients that received CD34⫺flt3⫺ cells could be repopulated with a higher
secondary repopulating rate (Fig. 4). These results demonstrated that CD34⫺flt3⫺ SRCs have more potent secondary
reconstituting abilities in comparison with the other two
types of SRCs, and could sustain long-term human hematopoiesis in NOD/SCID mice.

SRC Activity of Culture-Generated CD34ⴙflt3ⴙ/ⴚCells by IBMI
Recently, we reported that our identified CD34⫺ SRCs could
produce CD34⫹ SRCs after being cocultured with the murine
stromal cell line HESS-5 [29]. Therefore, we cocultured
CD34⫺flt3⫺ cells with HESS-5 in the presence of SCF, TPO,
IL-3, IL-6, and G-CSF, as reported previously [29]. After 1
week, significant numbers of CD34⫹flt3⫺ and CD34⫹flt3⫹
cells were generated, as shown in Figure 5C. We then sorted
these two populations (CD34⫹flt3⫹/⫺ cells) and tested their
SRC activities by IBMI. Seven of 10 and 5 of 10 mice thatreceived either CD34⫹flt3⫹ or CD34⫹flt3⫺ cells were repopulated with human cells. (Table 2). Human cell repopulation rates
in mice that received transplants of either CD34⫹flt3⫹ orCD34⫹flt3⫺ cells were 1.2%– 8.8% (median, 4.5%) and 1.4%–
7.8% (median, 3.7%), respectively.

DISCUSSION
A number of studies have demonstrated that flt3 tyrosine kinase
receptor plays a pivotal role in the regulation of primitive
murine [1–10] and human [11–17, 19, 20] hematopoietic stem/
progenitor cells such as c-kit. In the murine model, the expression and functional significance of flt3 and c-kit receptors inearly hematopoiesis has been investigated extensively [1–10,
23–28, 40 – 42]. Many studies have suggested that murine plu-

Table 2. Severe combined immunodeficiency (SCID) repopulatingcell activity of culture-generated CD34⫹flt3⫹/⫺ cells

Sorted cell fraction

No. of cells
transplanted

CD34⫺fit3⫹ cells 1 ⫻ 104 to 2 ⫻ 104
CD34⫹flt3⫺ cells 2 ⫻ 104 to 4 ⫻ 104

Human CD45ⴙ/cells (%)
Engraftment Range Median

7/10
5/10

1.2–8.8
1.4–7.8

4.5
3.7

Designated numbers of culture-generated CD34⫹fit3⫹ andCD34⫹flt3⫺ cells obtained by cell sorting were transplanted tononobese diabetic/SCID mice by intra-bone marrow (BM)injection. After 8 weeks, repopulation of human CD45⫹ cells inmurine BMs was analyzed.

ripotent long-term repopulating hematopoietic stem cells (LTRHSCs) express c-kit [40 – 42]. Particularly, Osawa et al. [23]clearly indicated that a single CD34⫺ KSL cell efficiently
reconstituted as many as one of five recipient mice. However,they did not rule out the possibility of coexistence of a lessfrequent c-kit⫺ LTR-HSC, which might fail to home into the
BM niche by intravenous injection. In support of the potential
existence of c-kit⫺ murine HSCs, several studies have reported
the existence of LTR-HSCs with little or no c-kit expression[43– 45]. Among them, Doi et al. [43] clearly demonstrated thatcandidate HSC could be subdivided into c-kitlow and c-kit⬍low(no detectable cell surface expression but positive for mRNA
expression) populations. Both populations could support donortype long-term multilineage reconstitution in primary recipients.
However, only c-kit⬍low HSCs showed secondary and tertiaryreconstituting capacity. In addition, Ortiz et al. [44] reported thatc-kit⫺ pluripotent stem cells can give rise to c-kit⫹ cells withcolony-forming unit in spleen (CFU-S) activity, suggesting thatc-kit⫹ HSCs are recruited from a more primitive quiescentc-kit⫺ HSC population. Collectively, these reported studies suggest that most of the murine LTR-HSCs express a low to highlevel of c-kit on their surfaces, but also that there is a lessfrequent subpopulation expressing less than a low level of c-kit
coexisting in murine BMs.
On the other hand, the flt3 receptor has also been shown to
be expressed and to function in murine candidate HSCs [1–10,
24 –28], including Lin⫺Sca-1⫹AA4⫹ fetal liver cells [5],CD34⫹/⫺ KSL BM cells [10, 24 –26], and Thy-1.1loKLS cells[9]. In particular, Jacobsen et al. have extensively studied theexpression and functional significance of flt3 receptor on mu-

Downloaded from www.StemCells.com at Swets Information Services on November 19, 2007

Figure 4. Secondary repopulating capacities of Lin⫺CD34⫹flt3⫹/⫺ or
Lin⫺CD34⫺flt3⫺ cells. Cells transplanted to primary recipients (PRs)by intra-BM injection (IBMI) numbered 3 ⫻ 103 to 5 ⫻ 103
CD34⫹flt3⫹cells, 4 ⫻ 103 to 5 ⫻ 103 CD34⫹flt3⫺ cells, or 2 ⫻ 104 to
3 ⫻ 104 CD34⫺flt3⫺ cells. Human cell repopulations of BMs in PRs(open circles) analyzed 8 –12 weeks after transplantation were comparable and 4%– 8%. Whole BM cells obtained from PRs were transplanted to secondary recipients (SRs) by IBMI. Human cell repopulationin SRs (closed circles) was analyzed 8 –10 weeks after secondarytransplantation. Horizontal bars represent each median of the repopulation rates in PRs and SRs, respectively. Abbreviation: BM, bone marrow.

Figure 5. Expression pattern of CD34 and flt3 on sorted
Lin⫺CD34⫺flt3⫺ cells after the 7-day coculture with HESS-5 cells. (A):Flow cytometry pattern of immunomagnetically separated cord bloodderived Lin⫺ cells stained with anti-flt3 (PE) and anti-CD34 (PC5)monoclonal antibodies. Lin⫺CD34⫺flt3⫺ cells were sorted for the coculture with HESS-5 cells. The sorting gate is indicated by the solid
square. (B): Postsorting analysis of the sorted Lin⫺CD34⫺flt3⫺ cells.
(C): The expression pattern of flt3 on CD34⫹ cells derived from the7-day cocultures of sorted Lin⫺CD34⫺flt3⫺ cells with the murine
stromal cell line, HESS-5, in the presence of a cocktail of cytokines. The
sorting gates for culture-generated CD34⫹flt3⫹/⫺ cells are indicated by
two solid squares. Abbreviation: PE, phycoerythrin.

Kimura, Asada, Wang et al.

www.StemCells.com

press flt3, and that the expression pattern of flt3 and c-kit
receptors on primitive mouse and human HSCs is different
and contrasting. However, they did not investigate the secondary repopulating capacity of CD34⫹flt3⫹/⫺ cells as wellas the repopulation capacity of the CD34⫺ counterpart.
In this study, we have investigated the SRC activity ofCB-derived
Lin⫺CD34⫹flt3⫹/⫺
cells
as
well
as
Lin⫺CD34⫺flt3⫹/⫺ cells using the IBMI method. First, we
confirmed that CB-derived Lin⫺CD34⫹flt3⫹/⫺ cells showeddistinct SRC activity by IBMI (Fig. 3; Table 1). Interestingly,
we demonstrated for the first time that Lin⫺CD34⫺flt3⫺ cells
showed significant and potent SRC activity by IBMI. Moreover,
our secondary transplantation study clearly indicated that thesecondary repopulating capacity is most potently observed in
CD34⫺flt3⫺ cells in comparison with CD34⫹flt3⫹/⫺ cells (Fig.
4). Finally, we observed that these Lin⫺CD34⫺flt3⫺ cells could
produce CD34⫹flt3⫹/⫺ SRCs after being cocultured with
HESS-5 cells in the presence of a cocktail of cytokines (Fig. 5).
These results suggest that CD34⫺flt3⫺ SRCs are the precursor
for CD34⫹flt3⫹/⫺ SRCs. On the basis of the results of ourpresent study, we propose that the immunophenotype of very
primitive human LTR-HSCs is Lin⫺CD34⫺c-kit⫺flt3⫺. Primitive human LTR-HSCs may express lower levels of c-kit andflt3 receptors on their surfaces when they commit to moremature short-term repopulating HSCs (STR-HSCs). It is still
unclear whether such a distinct pattern of c-kit and flt3 expression might identify distinct subpopulations of LTR-HSC or
STR-HSC within the human HSC hierarchy.
From another point of view, we and many other investigators have planned to expand candidate human HSCs ex vivousing several cytokines, including SCF, FL, TPO, and IL-6/
soluble IL-6 receptor (or fusion protein) [53–56]. However, thepresent study and other reported studies [24 –26, 41– 43,49 –52]
have demonstrated/suggested that very primitive LTR-HSCs do
not express their receptors, such as c-kit and flt3. Furthermore,
Lin⫺CD34⫺c-kit⫺flt3⫺ cells are still heterogeneous, and putative human LTR-HSC may express other potentially important
stem cell molecules. Therefore, for clinical application in thenear future, further studies will be required to elucidate theproposed model of the human HSC hierarchy as well as to
identify hitherto unidentified molecules that are important (indispensable) for stem cell expansion.
In conclusion, the present study provides evidence thathuman CB-derived CD34⫺ SRCs do not express flt3 tyrosine
kinase receptors, like murine candidate HSCs CD34⫺ KSL
cells. According to our data, the immunophenotype of human
LTR-HSC is Lin⫺CD34⫺c-kit⫺flt3⫺. Therefore, further studies
will be required to identify positive markers, such as Sca-1 formurine CD34⫺ KSL cells, for these primitive human LTRHSCs in the near future.

ACKNOWLEDGMENTS
We thank Dr. Takeshi Todo of Department of Mutagenesis,
Radiation Biology Center, Kyoto University, for his advice on
the irradiation of NOD/SCID mice; Kirin Brewery Co. Ltd.
(Tokyo) and Ajinomoto Co. Inc. (Yokohama, Japan) for providing the various growth factors used in this study; and Yuko
Masai for assistance in preparation of the manuscript. This work
was supported by Grants-in-Aid for Scientific Research on
Priority Areas (Grant number 15039227) and for Scientific
Research C (Grant number 15591015) 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 grantDownloaded from www.StemCells.com at Swets Information Services on November 19, 2007

rine LTR-HSCs [7, 8, 10, 24 –26]. Adolfsson et al. [24] firstreported that the upregulation of flt3 on BM-derived KSL cells
is accompanied by loss of self-renewal capacity. In other words,
flt3⫹ KSL cells rapidly and efficiently reconstituted B and T
lymphopoiesis, and only flt3⫺ KSL cells supported sustained
multilineage reconstitution [24 –26]. On the basis of these data,
they proposed that flt3⫹ KSL cells are progenitors for thecommon lymphoid progenitor (CLP) [24 –26]. Earlier, Kondo etal. [46] identified other CLPs that have the Lin⫺Thy-1⫺Sca1lowc-kitlowIL-7R␣⫹ immunophenotype. In Adolfsson’s study[25], it was shown that the flt3⫹ KSL cells produced thisIL-7R␣⫹ CLP in vitro as well as in vivo. These results suggested that the flt3⫹ KSL population is distinct and most likely
an intermediate between flt3⫺ KSL (LTR-HSC) and this IL7R␣⫹ CLP. Interestingly, flt3⫹ KSL cells were found to bealmost exclusively CD34⫹, whereas flt3⫺ KSL cells contained
a small but significant (5%) fraction of CD34⫺ cells [24]. This
notion was further supported by the recent study reported by
Sitnicka et al. [26]. In this study, FL-deficient mice had severely(10-fold) reduced levels of CLP, although the numbers ofcommon myeloid progenitors (CMPs) and CD34⫺ KSL were
unaffected [26]. Very recently, Adolfsson et al. [25] haveclearly demonstrated that the herein-mentioned flt3⫹ KSL
cells sustain granulocyte, monocyte, and B- and T-cell potential, but fail to produce significant erythroid andmegakaryocytic progeny. On the basis of these observations,they proposed an alternative road map for adult mouse blood
lineage commitment [25].
In contrast to murine LTR-HSC, the expression and functional significance of flt3 and c-kit on human LTR-HSC has yet
to be fully elucidated. Earlier studies have shown that most, if
not all, long-term culture-initiating cell (LTC-IC) are c-kit⫹ [47,48]. However, we observed that extended LTC-IC (ELTC-IC;
assayed after 7–9 weeks of coculture with allogeneic BM stromal layer) are apparently enriched in a CB-derived CD34⫹ckitlow/⫺ cell population [21]. Of note was that ELTC-ICs assayed after 9 weeks of coculture were detected only in the
CD34⫹c-kit⫺ cell population [21]. Our data are consistent withseveral other in vitro studies [49 –51]. Sogo et al. [49] haveclearly demonstrated that CB-derived CD34⫹c-kit⬍low cells mature into CD34⫹c-kitlow and CD34⫹c-kit⫹ cells in vitro, suggesting that the upregulation of c-kit protein on c-kit⬍low cells isthe first maturational step of human HSCs [49]. Enrichment of
human BM-derived primitive HSCs in the CD34⫹c-kitlow fraction was also confirmed using long-term engraftment studies inpreimmune fetal sheep [51]. The findings showed that BMderived CD34⫹c-kitlow cells transplanted to fetal sheep sustained
long-term donor-derived hematopoiesis (up to 16 months) [52].
There is, however, no direct evidence yet for a distinct populationof c-kit⫺ or c-kit⬍low human primitive HSCs with long-term repopulating potential. Such a stem cell population is likely to bepresent at very low frequency in human BM- or CB-derived hematopoietic cells. Recently, we identified very primitive CD34⫺
SRCs in human CB detected only by the IBMI method [29, 30]. As
shown in Figure 1D, the CB-derived Lin⫺CD34⫺ cell population
did not express detectable levels of c-kit protein by flow cytometry.
Therefore, our identified CD34⫺ SRCs may correspond to suchc-kit⫺ or c-kit⬍low LTR-HSCs [29, 30].
In contrast to c-kit, the information regarding flt3 expression on human LTR-HSCs is much more limited. Recently,
Sitnicka et al. [31], using the conventional intravenous injection method, clearly demonstrated that human BM- or
CB-derived CD34⫹ HSC capable of multilineage engrafting
NOD/SCID mice do express flt3 receptors. Moreover, theyalso showed that CB-derived CD34⫹flt3⫺ cells could repopulate recipient mouse BMs. On the basis of these data, they
proposed that most BM- and CB-derived CD34⫹ SRCs ex-

1353

SRC Activity of CD34⫺Flt3⫺ Cells by IBMI

1354

from 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 grantfrom Kansai Medical University (Research Grant B), a grantfrom the Japan Leukemia Research Foundation, and a grantfrom the Mitsubishi Pharma Research Foundation.

REFERENCES
1

The authors indicate no potential conflicts of interest.

long-term culture-initiating cells and telomerase expression. Leukemia
1998;12:728 –734.
22 Krause DS, Fackler MJ, Civin CI et al. CD34: Structure, biology, andclinical utility. Blood 1996;87:1–13.
23 Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic
reconstitution by a single CD34-low/negative hematopoietic cell. Science 1996;273:242–245.
24 Adolfsson J, Borge OJ, Bryder D et al. Upregulation of flt3 expression
within the bone marrow Lin⫺Sca1⫹c-kit⫹ stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 2001;15:659 – 669.
25 Adolfsson J, Mansson R, Buza-Vidas N et al. Identification of flt3⫹
lympho-myeloid stem cells lacking erythro-megakaryocytic potential: Arevised road map for adult blood lineage commitment. Cell 2005;121:
295–306.
26 Sitnicka E, Bryder D, Theilgaard-Monch K et al. Key role of flt3 ligand
in regulation of the common lymphoid progenitor but not in maintenanceof the hematopoietic stem cell pool. Immunity 2002;17:463– 472.
27 Mackarehtschian K, Hardin JD, Moore KA et al. Targeted disruption of
the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 1995;3:147–161.
28 McKenna HJ, Socking KL, Miller RE et al. Mice lacking flt3 ligand have
deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 2000;95:3489 –3497.
29 Wang J, Kimura T, Asada R et al. SCID-repopulating cell activity ofhuman cord blood-derived CD34⫺ cells assured by intra-bone marrow
injection. Blood 2003;101:2924 –2931.
30 Kimura T, Wang J, Matsui K et al. Proliferative and migratory potentials
of human cord blood-derived CD34⫺ severe combined immunodeficiency repopulating cells that retain secondary reconstituting capacity.
Int J Hematol 2004;79:328 –333.
31 Sitnicka E, Buza-Vidas N, Larsson S et al. Human CD34⫹ hematopoietic
stem cells capable of multilineage engrafting NOD/SCID mice express
flt3: Distinct flt3 and c-kit expression and response patterns on mouseand candidate human hematopoietic stem cell. Blood 2003;102:881– 886.
32 Ebihara Y, Wada M, Ueda T et al. Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice byclonal cells expanded from single CD34⫹CD38⫺ cells expressing flk2/
flt3. Br J Haematol 2002;119:525–534.
33 Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of
signals through gp130, c-kit, and interleukin-3 receptor promotes atrilineage blood cell production in the absence of terminally acting
lineage-specific factors. Blood 1997;90:4767– 4778.
34 Kimura T, Wang J, Minamiguchi H et al. Signal through gp130 activatedby soluble interleukin (IL)-6 receptor(R) and IL-6 or IL-6R/IL-6 fusion
protein enhances ex vivo expansion of human peripheral blood-derived
hematopoietic progenitors. STEM CELLS 2000;18:444 – 452.
35 Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection ofallogeneic bone marrow cells: A powerful new strategy for treatment of
intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:
3292–3299.
36 Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive
human hematopoietic cells capable of repopulating NOD/SCID mouse bonemarrow: Implications for gene therapy. Nat Med 1996;2:1329 –1337.
37 Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class ofhuman hematopoietic cells with SCID-repopulating activity. Nat Med
1998;4:1038 –1045.
38 Ogata K, Satoh C, Tachibana M et al. Identification and hematopoietic
potential of CD45⫺ clonal cells with very immature phenotype(CD45⫺CD34⫺CD38⫺Lin⫺) in patients with myelodysplastic syndromes. STEM CELLS 2005;23:619 – 630.
39 Tsuji T, Ogasawara H, Aoki Y et al. Characterization of murine stromal
cell clones established from bone marrow and spleen. Leukemia 1996;
10:803– 812.
40 Osawa M, Nakamura K, Nishi N et al. In vivo self-renewal of c-kit⫹Sca1⫹Linlow/⫺ hemopoietic stem cells. J Immunol 1996;156:3207–3214.
41 Ogawa M, Matsuzaki Y, Nishikawa S et al. Expression and function ofc-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63–71.
42 Orlic D, Fischer R, Nishikawa S et al. Purification and characterizationof heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 1993;82:762–770.
43 Doi H, Inaba M, Yamamoto Y et al. Pluripotent hemopoietic stem cells
are c-kit⬍low. Proc Natl Acad Sci U S A 1997;94:2513–2517.
44 Ortiz M, Wine JW, Lohrey N et al. Functional characterization of a novel

Downloaded from www.StemCells.com at Swets Information Services on November 19, 2007

Lyman SD, Jacobsen SEW. c-kit ligand and flt3 ligand: Stem/progenitor
cell factors with overlapping yet distinct activities. Blood 1998;91:
1101–1134.
2 Matthews W, Jordan CT, Wiegand GW et al. A receptor tyrosine kinase
specific to hematopoietic stem and progenitor cell-enriched populations.
Cell 1991;65:1143–1152.
3 Lyman SD, James L, Bos TV et al. Molecular cloning of a ligand for the
flt3/flk2 tyrosine kinase receptor: A proliferative factor for primitive
hematopoietic cells. Cell 1993;75:1157–1167.
4 Hannum C, Culpepper J, Cambell D et al. Ligand for flt3/flk2 receptor
tyrosine kinase regulates growth of haematopoietic stem cells and is
encoded by variant RNAs. Nature 1994;368:643– 648.
5 Zeigler FC, Bennett B, Jordan CT et al. Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells. Blood 1994;84:2422–2430.
6 Hirayama F, Lyman SD, Clark SC et al. The flt3 ligand supports
proliferation of lymphohematopoietic progenitors and early B-lymphoidprogenitors. Blood 1995;85:1762–1768.
7 Jacobsen SEW, Okkenhaug C, Myklebust J et al. The flt3 ligand potently
and directly stimulates the growth and expansion of primitive murinebone marrow progenitor cells in vitro: Synergistic interactions withinterleukin (IL) 11, IL-12, and other hematopoietic growth factor. J Exp
Med 1995;181:1357–1363.
8 Veiby OP, Jacobsen FW, Cui L et al. The flt3 ligand promotes thesurvival of primitive hematopoietic progenitor cells with myeloid as wellas B lymphoid potential. J Immunol 1996;157:2953–2960.
9 Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem
cell differentiation: A simple method to isolate long-term stem cells. Proc
Natl Acad Sci U S A 2001;98:14541–14546.
10 Yang L, Bryder D, Adolfsson J et al. Identification of LinSca1⫹kit⫹CD34⫹Flt3⫺ short-term hematopoietic stem cells capable of
rapidly reconstituting and rescuing myeloablated transplant recipients.
Blood 2005;105:2717–2723.
11 Lyman SD, James L, Johnson L et al. Cloning of the human homologueof the murine flt3 ligand: A growth factor for early hematopoietic
progenitor cells. Blood 1994;83:2795–2801.
12 Muench MO, Roncarolo MG, Menon S et al. Flk-2/flt-3 ligand regulates
the growth of early myeloid progenitors isolated from human fetal liver.
Blood 1995;85:963–972.
13 Shah AJ, Smogorzewska EM, Hannum C et al. Flt3 ligand induces
proliferation of quiescent human bone marrow CD34⫹CD38⫺ cells andmaintains progenitor cells in vitro. Blood 1996;87:3563–3570.
14 Rusten LS, Lyman SD, Veiby OP et al. The flt3 ligand is a direct andpotent stimulator of the growth of primitive and committed human
CD34⫹ bone marrow progenitor cells in vitro. Blood 1996;87:
1317–1325.
15 Rappold I, Ziegler BL, Kohler I et al. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing flt3
(CD135) receptor tyrosine kinase. Blood 1997;90:111–125.
16 Gotze KS, Ramirez M, Tabor K et al. Flt3high and flt3low CD34⫹
progenitor cells isolated from human bone marrow are functionallydistinct. Blood 1998;91:1947–1958.
17 Xiao M, Oppenlander BK, Plunkett JM et al. Expression of flt3 and c-kit
during growth and maturation of human CD34⫹CD38⫺ cells. Exp Hematol 1999;27:916 –927.
18 Sonoda Y, Sakabe H, Ohmizono Y et al. Synergistic actions of stem cell
factor and other burst-promoting activities on proliferation of CD34⫹
highly purified blood progenitors expressing HLA-DR or different levelsof c-kit protein. Blood 1994;84:4099 – 4106.
19 Sonoda Y, Kimura T, Sakabe H et al. Human flt3 ligand acts on myeloid
as well as multipotential progenitors derived from purified CD34⫹ blood
progenitors expressing different levels of c-kit protein. Eur J Haematol
1997;58:257–264.
20 Sakabe H, Kimura T, Zeng ZZ et al. Haematopoietic action of flt3 ligand
on cord blood-derived CD34-positive cells expressing different levels of
flt3 or c-kit tyrosine kinase receptor: Comparison with stem cell factor.
Eur J Haematol 1998;60:297–306.
21 Sakabe H, Yahata N, Kimura T et al. Human cord blood-derived primitive progenitors are enriched in CD34⫹c-kit⫺ cells: Correlation between

DISCLOSURE OF POTENTIAL CONFLICTS
OF INTEREST

Kimura, Asada, Wang et al.

www.StemCells.com

51 Laver JH, Abboud MR, Kawashima I et al. Characterization of c-kit
expression by primitive hematopoietic progenitors in umbilical cord
blood. Exp Hematol 1995;23:1515–1519.
52 Kawashima I, Zanjani ED, Almaida-Porada G et al. CD34⫹ humanmarrow cells that express low levels of kit protein are enriched forlong-term marrow-engrafting cells. Blood 1996;87:4136 – 4142.
53 Ohmizono Y, Sakabe H, Kimura T et al. Thrombopoietin augments ex
vivo expansion of human cord blood-derived hematopoietic progenitors
in combination with stem cell factor and flt3 ligand. Leukemia 1997;11:524 –530.
54 Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo
culture. J Exp Med 1997;186:619 – 624.
55 Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative
assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997;94:9836 –9841.
56 Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCIDrepopulating cells by stem cell factor, flk2/flt3 ligand, thrombopoietin,
IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105:1013–1021.

Downloaded from www.StemCells.com at Swets Information Services on November 19, 2007

hematopoietic stem cell and its place in the c-kit maturation pathway inbone marrow cell development. Immunity 1999;10:173–182.
45 Randall TD, Weissman IL. Characterization of a population of cells in thebone marrow that phenotypically mimics hematopoietic stem cells: Restingstem cells or mystery population? STEM CELLS 1998;16:38 – 48.
46 Kondo M, Weissman IL, Akashi K. Ientification of clonogenic common
lymphoid progenitors in mouse bone marrow. Cell 1997;91:661– 672.
47 Simmons PJ, Aylett GW, Niutta S et al. C-kit is expressed by primitive
human hematopoietic cells that give rise to colony-forming cells on
stroma-dependent or cytokine-supplemented culture. Exp Hematol 1994;
22:157–165.
48 Briddell RA, Broudy VC, Bruno E et al. Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a
monoclonal antibody to the c-kit receptor. Blood 1992;79:3159 –3167.
49 Sogo S, Inaba M, Ogata H et al. Induction of c-kit molecules on human
CD34⫹/c-kit⬍low cells: Evidence for CD34⫹/c-kit⬍low cells as primitive
hematopoietic stem cells. STEM CELLS 1997;15:420 – 429.
50 Gunji Y, Nakamura M, Osawa H et al. Human primitive hematopoietic
progenitor cells are more enriched in KITlow cells than in KIThigh cells.
Blood 1993;82:3283–3289.

1355

Identification of Long-Term Repopulating Potential of Human Cord
Blood-Derived CD34 –flt3– Severe Combined Immunodeficiency-Repopulating
Cells by Intra-Bone Marrow Injection
Takafumi Kimura, Rumiko Asada, Jianfeng Wang, Takashi Kimura, Miho Morioka,
Kazuo Matsui, Katsuya Kobayashi, Kae Henmi, Shiro Imai, Masakazu Kita, Takashi
Tsuji, Yutaka Sasaki, Susumu Ikehara and Yoshiaki Sonoda
Stem Cells 2007;25;1348-1355; originally published online Feb 15, 2007;
DOI: 10.1634/stemcells.2006-0727
This information is current as of November 19, 2007
including high-resolution figures, can be found at:
http://www.StemCells.com/cgi/content/full/25/6/1348

Downloaded from www.StemCells.com at Swets Information Services on November 19, 2007

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