Experimental Hematology 2011;39:203–213

Development of a high-resolution purification method
for precise functional characterization of primitive human
cord blood–derived CD34–negative SCID-repopulating cells
Mari Ishiia,d, Yoshikazu Matsuokaa, Yutaka Sasakia, Ryusuke Nakatsukaa, Masaya Takahashia,b,Tsuyoshi Nakamotoc, Katsuhiko Yasudac, Kazuo Matsuie, Hiroaki Asanof, Yasushi Uemurag,Takashi Tsujih, Shirou Fukuharad, and Yoshiaki SonodaaaDepartment of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science; bDepartment of Pediatrics; cDepartment of
Obstetrics and Gynecology; dFirst Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan; eDepartment of
Gynecology and Obstetrics, Fukuda Hospital, Kumamoto, Japan; fSchool of Nursing, Kyoto Prefectural University of Medicine, Kyoto, Japan;gDivision of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan; hDepartment of Biological Science and Technology, Faculty ofIndustrial Science and Technology, Tokyo University of Science, Chiba, Japan(Received 16 September 2010; revised 17 November 2010; accepted 17 November 2010)

Objective. We have successfully identified human cord blood (CB)–derived CD34-negative(CD34L) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive
lymphomyeloid repopulating ability using the intrabone marrow injection method. In ourprevious study, a limiting dilution analysis demonstrated the frequency of CD34L SRCs in CBderived 13lineage-negative (LinL) CD34L cells to be approximately 1/25,000. In this study, we
intended to develop a high-resolution purification method to obtain highly purified CD34L SRCs.
Materials and Methods. The pooled CB-derived LinL cells were stained with 13 reported Lin
monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA,
and CD127. Then 18LinLCD34high, 18LinLCD34L, and 13LinLCD34highCD38L cells were
sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractionsof cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of thisnew purification method.
Results. A limiting dilution analysis demonstrated the frequency of CD34L SRCs in these
18LinLCD34L cells to be approximately 1/1,000, which is associated with a seeding efficiency
25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/
IL-2Rgcnull mice that received transplants of only two CD34L SRCs were highly engraftedwith human lymphomyeloid cells at 24 weeks after primary transplantation and showedsecondary multilineage repopulating abilities.
Conclusions. We succeeded to highly purify the CD34L SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification
method is indispensable to precisely characterize a distinct class of primitive human CBderived CD34L hematopoietic stem cells. Ó 2011 ISEH - Society for Hematology andStem Cells. Published by Elsevier Inc.

Hematopoietic stem cells (HSCs) are a self-renewing population with the developmental potential to give rise to allof the mature blood cell types [1–3]. Recent advances in
Offprint requests to: Yoshiaki Sonoda, Ph.D., Department of Stem Cell
Biology and Regenerative Medicine, Graduate School of Medical Science,
Kansai Medical University, Moriguchi, Osaka, 570-8506, Japan; E-mail:sonoda@takii.kmu.ac.jp
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.exphem.2010.11.008.

fluorescence-activated cell sorting (FACS) technology
make it possible to prospectively isolate HSCs to a highpurity using various cell surface markers. Among them,
the CD34 antigen (Ag) has long been believed to be a reliable HSC marker in mammals, including mice and humans[4]. However, it was reported that murine bone marrow(BM)–derived long-term lymphomyeloid reconstituting
HSCs are lineage marker–negative (Lin), c-kit–positive(c-kitþ), Sca-1–positive (Sca-1þ), and CD34-negative(CD34) cells, also known as CD34 KSL cells [5].

0301-472X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
doi: 10.1016/j.exphem.2010.11.008

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M. Ishii et al./ Experimental Hematology 2011;39:203–213

The measurement of the repopulation and differentiation
capabilities of primitive human HSCs has been greatly
facilitated by development of a xenotransplantation assay
system using severe combined immunodeficient (SCID)
mice, the SCID-repopulating cell (SRC) assay [6–8]. We
developed a highly sensitive SRC assay system using intrabone marrow injection (IBMI) technique [9]. Using thistechnique, we have successfully identified human cord
blood (CB)–derived CD34 SRCs with extensive lymphoidand myeloid repopulating abilities [9]. These CD34 SRCs
seemed to be more primitive HSCs than previously reported
CD34þ SRCs [9–13]. They could home into the BM nicheby IBMI only, partly because they expressed lower levels of
homing receptors, including CXCR4 [9–13]. Flow cytometry (FCM) revealed that the CD34 SRCs did not express
detectable levels of c-kit tyrosine kinase receptor [11], in
contrast to the expression observed in murine CD34
KSL cells [5]. In our previous studies [9–12], 13 lineage(Lin)-specific monoclonal antibodies (mAbs) against
CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19,
CD20, CD24, CD41, CD56, and CD235a, were used topurify CD34 SRCs. An in vivo limiting dilution analysis
using the IBMI demonstrated the frequency of the CD34
SRCs in the 13LinCD34 cells to be approximately
1/25,000 [12]. Conversely, the frequency of the SRCs in
CB-derived 13LinCD34þCD38 cells was 1/40 cells[12]. When these LinCD34þCD38 cells were transplanted by tail vein injection, the SRC frequency decreased
to approximately 1/600 cells [14,15]. These results demonstrated that the IBMI technique is associated with a seeding
efficiency 15 times greater than the tail vein injection.
Therefore, the SRC assay system using the IBMI technique
is very useful for investigating the HSC activity of certain
target populations. However, the SRC frequency in
13LinCD34 cells was still very low in comparison to
the frequency of SRC in 13LinCD34þCD38 cells.
Therefore, it is crucial to further purify the CD34 SRCs
in order to more precisely characterize the CD34 SRCs.
Here we report the development of a high-resolution
purification method for the rare class of primitive human
CD34 SRCs (HSCs) using mAbs to 18Lin-specific Ags,
including the 13 Ags and 5 additional Ags mentioned previously (CD11b, CD33, CD45RA, CD66c, and CD127).
Using these 18Lin mAbs, we have succeeded to highly
purify the human CB-derived CD34 SRCs.

Materials and methods
Collection of CB samples and processing of lineage-negativecells
CB samples were obtained from normal full-term deliveries withsigned informed consent. This study was approved by the Institutional Review Board of Kansai Medical University. The CBderived Lin mononuclear cells were separated using a StemSep
device (StemCell Technologies, Vancouver, BC, Canada) [9–12].

High-resolution purification of LinCD34 cells
The pooled Lin cells from multiple donors were stained with thefollowing: 7-amino-actinomycin D (7-AAD; Beckman Coulter,
Fullerton, CA, USA); fluorescein isothiocyanate (FITC)–
conjugated 13Lin mAbs, against CD2, CD16, CD24, and CD235a(DAKO, Kyoto, Japan), CD3, CD10, CD20, and CD41 (Beckman
Coulter), CD7, CD19, and CD56 (BD Biosciences, San Jose, CA,USA), CD4 and CD14 (eBioscience, San Diego, CA, USA);phycoerythrin (PE)-conjugated 5 Lin mAbs, against CD11b (Serotec, Oxford, UK), CD33 and CD66c (Beckman Coulter), CD45RA(Southern Biotech, Birmingham, AL, USA), and CD127 (R&D
Systems, Minneapolis, MN, USA); Pacific Blue–conjugated antiCD45 mAb (BioLegend, San Diego, CA, USA); apophycocyanin(APC)-conjugated anti-CD34 mAb (BD Biosciences); andphycoerythrin-cyanin 7 (PE-Cy7)-conjugated anti-CD38 mAb(BioLegend), as reported previously [9–12]. The stained cells
were then sorted into three fractions (18LinCD34high,
18LinCD34, and 13LinCD34highCD38 cells) using an FACS
Aria (BD Biosciences) (Fig. 1). A portion of the cells were cytospun and then stained with May-Gr€
unwald/Giemsa for themorphological analysis.
RNA preparation and reverse transcriptase polymerase chain
reaction (RT-PCR)
Total RNAs were isolated from 10,000 CB-derived 13Lin
CD34þCD38 or 18LinCD34 cells using RNeasy plus microkit (Qiagen, Valencia, CA, USA) according to manufacturer’s
instructions. The complementary DNAs were synthesized by using
the iScript DNA synthesis kit (Bio-Rad Laboratories, Hercules,
CA, USA). The complementary DNAs were amplified using AmpliTaq Gold DNA polymerase (Applied Biosystems, Tokyo,
Japan). PCR cycling protocol consisted of the following: 95 C
for 30 seconds; 58 C for 30 seconds; 72 C for 45 seconds; repeat
protocol for 38 cycles. The PCR products were separated byelectrophoresis on 1% agarose gels. The primer sets are listed in
Supplementary Table E1 (online only, available at www.exphem.
org).
Clonal cell culture
Colony-forming cells were assayed using our standard methylcellulose cultures as reported [11,16,17].
Coculture with HESS-5 cells and analysis
of culture-generated cells by FCM
A total of 1  103 purified 18LinCD34high and 1 to 3  103 purified 18LinCD34 cells per well of 24-well plate (BD Labware,Franklin Lakes, NJ, USA) were plated onto pre-established irradiated HESS-5 layers in StemPro-34 medium (Gibco Laboratories,
Grand Island, NY, USA); a cocktail of cytokines comprising 50
ng/mL stem cell factor (BioScience Autogen, Wiltshire, UK), 50
ng/mL flt3 ligand (R & D Systems), 100 ng/mL thrombopoietin(TPO), 10 ng/mL interleukin (IL)-3 (R&D Systems), 100 U/mL
IL-6 (kindly provided by Ajinomoto Co. Inc., Tokyo, Japan), 10
ng/mL granulocyte colony-stimulating factor (G-CSF), and 5%
fetal calf serum (HyClone, Laboratories, Logan, UT, USA). GCSF and TPO were generous gifts from Kyowa Kirin Company(Tokyo, Japan). After 1 week, all of the cells were collected byvigorous pipetting and a portion was used for cell counting using

M. Ishii et al./ Experimental Hematology 2011;39:203–213

205

Figure 1. Representative FACS profiles of CB-derived 13LinCD45þCD34highCD38 and 18LinCD45þCD34high/ cells. (A) The forward scatter/side
scatter (FSC/SSC) profile of immunomagnetically separated Lin cells. The R1 gate was set on the blast-lymphocyte window. (B) The R2 gate was set
on the living cells. (C, D) Expression of FITC-conjugated 13Lin markers, including CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24,
CD41, CD56, and CD235a, and the PE-conjugated 5 Lin markers, including CD11b, CD33, CD45RA, CD66c and CD127, are presented. (E) The
13Lin (R3) cells were subdivided into 2 fractions: 13LinCD45þCD34high (R5) and CD34 (R6) cells, according to their expression levels of CD34.
The definitions of CD34high/ cells are as follows: the CD34high fraction contains cells expressing maximum APC fluorescence intensity (FI) to 5% levelof FI and the CD34 fraction contains cells expressing !0.5% level of FI. (F) The 18Lin cells residing in the R4 gate were further subdivided into
two fractions: 18LinCD45þCD34high (R7) and CD34 (R8) cells, according to their expression levels of CD34. The definitions of CD34high/ cells are
as follows: the CD34high fraction contains cells expressing maximum APC FI to 15% level of FI and the CD34 fraction contains cells expressing !
0.2% level of FI. (G) The R5-gated cells were further subdivided into two fractions: 13LinCD45þCD34highCD38þ (R9) and CD38 (R10) cells. The definitions of CD38þ/ cells are as follows: the CD38þ fraction contains cells expressing maximum PE-Cy7 FI to 5% level of FI and the CD38 fraction contains
cells expressing !0.5% level of FI.

a counting chamber. The cells were then stained with 7-AAD, PEconjugated anti-CD34 mAb (Beckman Coulter) and APCconjugated anti-CD45 mAb (BD Biosciences). The number ofCD34þ cells was analyzed by FCM as described here.
NOD/SCID and NOG miceFemale 5-week-old nonobese diabetic (NOD)/Shi-scid/scid(NOD/SCID) mice were purchased from Clea Japan (Tokyo,
Japan). Female 6-week-old NOD/Shi-scid/IL-2Rgnull(NOG)cmice [18] were purchased from the Central Institute of Experimental Animals (Kawasaki, Japan). All mice were handled understerile conditions and were maintained 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.
IBMI of purified cells
IBMI was carried out as reported previously [9–12].

SRC assay and serial analysis of human CD45þ cellengraftment in NOD/SCID or NOG mice by FCM
SRC assays were performed using a previously reported method[9–12]. In this study, 400 to 2  103 purified CB-derived 18Lin
CD34 or 200 purified CB-derived 13LinCD34highCD38 cells
were transplanted by IBMI into sublethally irradiated (250 cGyusing a 137Cs-g irradiator) 7- to 8-week-old NOD/SCID or NOG
mice. The repopulation of human CD45þ hematopoietic cells in
NOD/SCID or NOG mouse BM was analyzed by FCM. The
mice were sacrificed 10 to 12 weeks after transplantation and
the BM cells were collected by crushing the pairs of femurs andtibiae of each mouse in a mortar. Cells were suspended in Ca2þand Mg2þ-free phosphate-buffered saline containing 2% fetal
calf serum and the remaining bone tissues were removed using
a cell strainer (BD Falcon). In long-term human hematopoietic
cell reconstitution experiments using NOG mice, 1 to 2 mL BM
fluid were serially aspirated from the contralateral sites (righttibia) of each mouse at 5, 8, 12, 18, and 24 weeks after206

M. Ishii et al./ Experimental Hematology 2011;39:203–213

transplantation and the BM fluid was then diluted with Ca2þ- andMg2þ-free phosphate-buffered saline containing 2% fetal calf
serum.
The repopulation of human hematopoietic cells in murine BM
was determined by detecting the number of 7-AAD cells positively stained with Pacific Blue–conjugated anti-human CD45
mAb by six-color FCM (FACS CantoII; BD Biosciences). The
mice were scored as positive if O0.1% of the total murine BM
cells were human CD45þ cells. Cells were also stained with PECy7–conjugated anti-mouse CD45.1 mAb (Beckman Coulter);PE-conjugated anti-human CD19 (eBioscience) and CD41 (Beckman Coulter) mAbs; FITC-conjugated anti-human CD33 andCD14 (eBiosciences), and anti-human CD235a mAbs (DAKO);
and APC-conjugated anti-human CD34 mAb (BD Biosciences)
for the detection of human stem/progenitor, lymphoid, andmyeloid hematopoietic cells.
For the precise analysis of human cell repopulation in NOG
mice at 24 weeks after transplantation, the NOG mice were sacrificed and then murine BM, peripheral blood (PB), and spleens
were collected. Cells obtained from these organs were stainedwith the previously mentioned anti-human mAbs, FITCconjugated anti-human CD3 and PE-conjugated anti-human CD4
mAbs (Beckman Coulter), and APC-conjugated anti-human CD8
mAb (eBioscience) to analyze the T-cell differentiation potentials.
In vivo limiting dilution analysis
To assess the frequency of SRCs in the CB-derived 18LinCD34
cells, various numbers of 18LinCD34 cells (400, 1000, and2000; n 5 20) were transplanted to NOD/SCID mice by IBMI

as reported [9–12]. The mice were sacrificed 10 to 12 weeks after
transplantation and the human cell repopulation in mouse BM wasanalyzed by FCM as described here.
Secondary transplantation
For secondary transplantations, murine BM cells were obtained 24
weeks after transplantation from the pairs of femurs and tibiae ofmoderately to highly engrafted primary recipient NOG micereceiving 2  103 18LinCD34 or 200 13LinCD34highCD38
cells. Three of the four portions of the whole BM cells were transplanted by IBMI into sublethally (250 cGy) irradiated secondary
recipient NOD/SCID mice. Twelve weeks after transplantation,
the presence of human CD45þ cells in the secondary recipients’
BM was analyzed by FCM, as described for primary transplantation.
Statistical analysis
In the limiting dilution analyses, the frequencies of SRCs were
calculated using the Poisson statistics as reported previously[9,10,12,14]. The significance of the differences in human cell repopulation in NOD/SCID or NOG mice was determined using theMann-Whitney U-test.

Results
High-resolution purification of human CB–derived
CD34 cells
First, the R1 gate was set on the blast-lymphocyte window(Fig. 1A). Next, the 7-AAD cells were gated as R-2 inFigure 1B. The 13LinCD45þ and 18LinCD45þ cells

Figure 2. Colony-forming capacities of sorted 18LinCD45þCD34high and CD34 cells.(A) Two hundred sorted CD34high and CD34 cells were cultured inmethylcellulose at 37 C with 5% CO2, 5% O2 and 90% N2 in the presence of stem cell factor, TPO, IL-3, GM-CSF, G-CSF, and erythropoietin for 12 to 14
days. Many macroscopic colonies containing erythroid cells are seen in both dishes. (B) The number and composition of various colonies derived from
CD34high and CD34 cells are shown. Open, shaded, and closed bars represent the number of CFU-Mix, BFU-E, and CFU-GM (including CFU-G,
CFU-M, and CFU-GM), respectively. The types of colonies identified in situ were granulocyte (CFU-G), macrophage (CFU-M), granulocyte/macrophage(CFU-GM), erythroid burst (BFU-E), and erythrocyte-containing mixed (CFU-Mix). The numbers of all the types of hematopoietic colonies were determinedas the mean of quintuple cultures.

M. Ishii et al./ Experimental Hematology 2011;39:203–213

were then gated as R3 and R4 (Fig. 1C, D), respectively. The
18LinCD45þ cells were further subdivided into two distinct
populations gated as R7 (CD34high) and R8 (CD34) basedon their surface CD34 expression (Fig. 1F). On the other
hand, the 13LinCD45þCD34high (R5) cells (Fig. 1E) were
further subdivided into two distinct populations gated as
R9 (CD34highCD38þ) and R10 (CD34highCD38) basedon their surface CD38 expression (Fig. 1G). Then
18LinCD34high (R7), 18LinCD34 (R8), and 13Lin
CD34highCD38 (R10) fractions were sorted for the subsequent in vitro cultures and in vivo SRC assays.

207

As shown in Figures 1E and F, the 18LinCD34 (R8)
fraction was highly purified (0.32% of R4 gated cells) incomparison to the 13LinCD34 (R6) fraction (3.63% of
R3 gated cells). The 13LinCD34highCD38 (R10) fraction
was 0.83% of R5 gated cells (Fig. 1G). Morphological analysis showed that almost all of the 18LinCD34 cells wereimmature blast-like in appearance, with high nuclear-tocytoplasmic ratio, fine chromatin network, a few nucleoli,
and a scant basophilic cytoplasm (data not shown).
Next, we analyzed the expression of various messenger
RNAs (mRNAs), including c-kit, flt3, c-Mpl, and IL-3Ra,

Figure 3. Coculture of 18LinCD34high and CD34 cells with HESS-5 cells. (A) 18LinCD34high and CD34 cells were cocultured with HESS-5 cells in
the presence of six cytokines (stem cell factor þ flt3 ligand þTPO þ IL-3 þ IL-6 þ G-CSF) for 1 week. Representative photomicrographs taken on day 7 arepresented. 18LinCD34 cells formed many large cobblestone areas (right panel). In contrast, 18LinCD34high cells mainly formed small cobblestone areas(left panel). (B) Expression patterns of CD34 antigen on the culture-generated CD45þ cells are shown. The percentages of CD34þ cells are presented in rightupper corner. (C) The number of CD34þ cells produced in coculture with HESS-5 cells is shown. The total number of cells derived from 1 to 3  103
18LinCD34 cells linearly expanded by 20-fold, and 30% to 40% of them consisted of CD34þ cells. On the other hand, the total number of cells derived
from 1  103 18LinCD34high cells expanded by 70-fold, and 60% to 70% of them consisted of CD34þ cells.

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M. Ishii et al./ Experimental Hematology 2011;39:203–213

of 18LinCD34 (R8) and 13LinCD34highCD38 (R10)cells by RT-PCR. As shown in Supplementary Figure E1(online only, available at www.exphem.org), both the
18LinCD34 and 13LinCD34highCD38 cells expressed
all of these mRNAs. However, the 18LinCD34 cells
weakly expressed the mRNAs of flt3, c-Mpl, and IL-3Ra
in comparison to those of 13LinCD34highCD38 cells.

Characteristics of hematopoietic colony-forming
capacity of CB-derived 18LinCD34highand CD34 cells
The colony-forming cell capacities of the CB-derived
18LinCD34high and CD34 cells were quite unique andmany macroscopic colonies were seen (Fig. 2A). The plating
efficiency of 18LinCD34high cell fraction was approximately 60% to 70%, which contained approximately 30%
colony-forming unit (CFU) granulocyte-macrophage (GM),60% burst-forming unit erythroid (BFU-E), and !10%
CFU-Mix (Fig. 2B). As previously reported [9], the13LinCD34 cell fraction showed almost no colony formation. In contrast, the plating efficiency of the 18LinCD34
cell fraction was 40% to 50%, which contained approximately !10% CFUGM, 60% BFU-E, and 40% CFUMix (Fig. 2B). These results demonstrated that the highly
purified 18LinCD34 cell fraction contains a large number
of BFU-E, as well as CFU-Mix, and also indicated for the firsttime that the colony-forming cell capacities are detected in
the CB-derived CD45þCD34 cell fraction.

Coculture with HESS-5 cells
The CD34 SRCs could produce CD34þ SRCs in vitro, asreported [9,11]. Therefore, 1 to 3  103 18LinCD34 and1  103 18LinCD34high cells were cocultured with themurine stromal cell line HESS-5 in the presence of cytokines (i.e., stem cell factor, flt3 ligand, TPO, IL-3, IL-6,
and G-CSF) for 1 week. The 18LinCD34 cells actively
proliferated and formed many large cobblestone areas(Fig. 3A, right panel). In contrast, the 18LinCD34high cells
mainly formed small cobblestone areas and many cells
were floating over the HESS-5 cells (Fig. 3A, left panel).
After 1 week, the cells in each well were collected byvigorous pipetting, and were then counted and analyzed
for the CD34þ cell content by FCM. The total number ofcells derived from 18LinCD34 cells expanded by 20fold, 30% to 40% of which consisted of CD34þ cells(Fig. 3B, C). A linear proliferation response was seen
with cells plated at 1 to 3  103 cells per well. On the other
hand, the total number of cells derived from 1  103
18LinCD34high cells expanded by about 70-fold, 60% to
70% of which consisted of CD34þ cells (Fig. 3B, C). These
results indicated that the 18LinCD34 cell fraction contained a substantial number of CD34þ cell-producing
hematopoietic progenitor cells/HSCs.

SRC activity and limiting dilution analysis
of CB-derived 18LinCD34 cells by IBMI
Thereafter, we investigated the SRC activity of the
18LinCD34 cells using NOD/SCID mice. As expected,
a limiting dilution analysis using the IBMI demonstrated
the frequency of the CD34 SRCs in the 18LinCD34
cells to be approximately 1/1000 (Fig. 4). The frequencyof the CD34 SRCs in the 13LinCD34 cells was 1/
25,000, as we reported previously [12]. These results clearly
demonstrated that application of the 18Lin mAbs plus the
IBMI technique is associated with a seeding efficiency
25 times greater than the previously described method.
Long-term repopulation patterns of CD34 SRCs
and CD34þCD38 SRCs in NOG mice
The next approach to characterize the self-renewal potential
and the long-term repopulating potential of the CD34
SRCs was to serially analyze the kinetics of BM engraftment for 24 weeks in NOG mice that received transplantsof 2  103 18LinCD34 cells (containing two SRCs).
This was compared with the repopulating patterns in
NOG mice that received 200 13LinCD34highCD38 cells(containing 5 SRCs) (Fig. 5). The numbers of SRCs transplanted were estimated based on the data obtained from thelimiting dilution analyses presented in Figure 4 andrecently reported data [12].
In these experiments, both the mice that received
transplants of CD34 and CD34þCD38 SRCs showed

Figure 4. The frequency of SRC in 18LinCD34 cells. Various numbersof 18LinCD34 cells (400, 1000, and 2000) were transplanted to NOD/SCID mice (n 5 20). The human CD45þ cell repopulation in the mouse
BM was analyzed by FCM 10 weeks after transplantation. The frequency
of SRC was 1 per 1021 18LinCD34 cells. For the frequency determination, the middle solid line represents the estimated weighted meanfrequency (fWM). The lower and upper dotted lines represent 95%
confidence interval of fWM.

M. Ishii et al./ Experimental Hematology 2011;39:203–213

209

Secondary repopulating ability of CD34 SRCs
and CD34þCD38 SRCs by IBMI
All of the secondary recipient mice that received the transplants from the primary recipient mice (receiving the transplants of two CD34 SRCs or five CD34þCD38 SRCs)
showed signs of human cell repopulation at 12 weeks after
transplantation (Table 1). The human CD45þ cell rates inall the secondary recipient mice showed comparable levelsof human cell repopulation (0.1% to 10.8% vs. 0.1% to10%). We confirmed that all of the secondary recipient micethat received transplants of CD34 as well as CD34þCD38
SRCs showed multilineage human hematopoiesis, including
CD34þ, CD19þ, and CD33þ cells (data not shown).
These results demonstrated that the CD34 andCD34þCD38 SRCs both had comparable levels of
secondary reconstituting abilities and could sustain longterm (up to 36 weeks) human hematopoiesis in NOG and
NOD/SCID mice, suggesting that both SRCs had significant self-renewing potential.

Figure 5. Long-term human hematopoietic cell reconstitution in NOG
mice. Five CD34þCD38 SRCs (dotted line) or 2 CD34 SRCs (solidline) were injected into the left tibiae of NOG mice by IBMI. The numberof SRCs transplanted was estimated based on the limiting dilution analysis
data. The human CD45þ cell rates in the contralateral sites of recipient
mice were serially analyzed by the BM aspiration method 5, 8, 12, 18,
and 24 weeks after transplantation by six-color FCM. Data represent themedian 6 standard deviation of results from six mice at each time point
except 8 weeks (n 5 3). *p ! 0.05.

Comparison of the differentiation potentials
of CB-derived CD34 SRCs and CD34þCD38 SRCs
To further evaluate the functional differences between the
CD34 and CD34þCD38 SRCs, we studied their multilineage reconstitution abilities in various organs, including
the BM, PB, and spleen, in NOG mice that were transplanted with either 2  103 18LinCD34 cells (2 SRCs)
or 200 13LinCD34highCD38 cells (5 SRCs) by IBMI.
The results of the BM repopulation patterns are shown in
Figure 6. Analyses of the two representative mice thatwere transplanted with either CD34 SRCs (Fig. 6, rightcolumn) or CD34þCD38 SRCs (Fig. 6, left column)
demonstrated that both of the CD34 and CD34þCD38
SRCs generated CD34þ stem/progenitor cells and both
SRCs had a comparable in vivo differentiation capacity to
the B-lymphoid, myeloid, monocytic, megakaryocytic,
and erythroid lineages 24 weeks after the transplantation.
Next, the percentages of T-lymphoid cells in variousorgans were analyzed (Fig. 7). The CD4þ and CD8þ single
positive T-lymphoid cells were detected in the CD3þ cellgates of the BM, PB, and spleen in both NOG mice thatwere transplanted with either CD34- SRCs or CD34þCD38SRCs. These results clearly demonstrated that the CD34
SRCs could supply comparable levels of maturesigns of human cell repopulation at 5 weeks after transplantation (Fig. 5). The repopulation level of all six mice thatreceived transplants of two CD34 SRCs was 0.4% to28.9% (median: 2.5%). Five of six mice that received transplants of five CD34þCD38 SRCs also showed a comparable level of human cell repopulation (0.2% to 28.7%;
median: 0.4%). The mice that received transplants of
CD34þCD38 SRCs showed a peak level of repopulation
at 18 weeks after transplantation (74.2% to 98.5%; median:
90.0%), and their repopulation rates were maintained at thissame level until 24 weeks after transplantation. On
the other hand, the repopulation rates of the mice thatreceived transplants of CD34 SRCs gradually increased
over 5 to 12 weeks, then reached high levels and maintained
the same levels (74.9% to 88.0%; median: 81.6% at 24
weeks after transplantation) during the observation period.
These results demonstrated for the first time that the
CD34 SRCs had potent human cell repopulation potentials, which was almost equivalent to that of the
CD34þCD38 SRCs.
Table 1. Secondary repopulating ability of CD34þCD38 and CD34 SRCs
Type and no. of cells
transplanted into PR

No. of
SRCs*% Humancells in PRy

No. of cells transplanted
into SR (107)

Incidence ofengraftment in SR% Humancells in SRz

CD34highCD38, 200
CD34, 2000

52

26.6–72.6 (67.0)
14.8–65.1 (50.6)

4.2–5.2 (4.5)
4.0–5.5 (5.2)

4/4
5/5

0.1–10.8 (0.76)
0.1–10 (1.01)

Median values are presented in parentheses.
PR 5 primary recipient; SR 5 secondary recipient.
*No. of SRCs transplanted is estimated by limiting dilution analysis.
yHuman cell repopulation of BM in PR NOG mice was analyzed 24 weeks after transplantation.
z
Human cell repopulation of BM in SR NOD/SCID mice was analyzed 12 weeks after secondary transplantation.

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T-lymphoid cells at 24 weeks after transplantation similar
to the CD34þCD38 SRCs.
Discussion
A number of studies, including ours, have demonstrated that
long-term– and short-term–repopulating SRCs are present
in human CB and BM cells [6–15,19–22] and also provided
evidence that CB- or BM-derived CD34þCD38 SRCs arevery primitive human HSCs [7,8,12,14,20–22]. As reported,
we first identified human CB-derived CD34 SRCs using
a sensitive IBMI method, which can detect these CD34
SRCs very efficiently [9]. Because these CD34 SRCs
expressed lower levels of homing receptors, including
CXCR4, they could not home into the BM niche byconventional tail vein injection methods [9]. In a series ofexperiments, we extensively studied the functional characteristics of the CD34 SRCs using in vitro cultures withmurine stromal HESS-5 cells as well as in vivo repopulation
assays using NOD/SCID or NOG mice [9–12]. Importantly,
the CD34 SRCs could produce CD34þ SRCs in vitro as
well as in vivo [9,11]. Moreover, the CB-derived CD34
SRCs did not express c-kit and flt3 tyrosine kinase receptors,
thereby suggesting that the surface immunophenotype ofhuman CD34 SRCs is LinCD34c-kitflt3 [11,13].
However, the 18LinCD34 cells weekly expressed themRNAs of c-kit and flt3 by RT-PCR (Supplementary
Figure E1; online only, available at www.exphem.org).
Except for the expression of c-kit, these findings are consistent with the earlier report of Adolfsson and colleagues [23]that very primitive murine long-term HSCs (CD34 KSL
cells) did not express flt3. We also reported that the CBderived CD34 SRCs could produce CD34þflt3 andCD34þflt3þ SRCs in vitro [11]. Collectively, the CD34
SRCs are a distinct class of the HSC population and appear
to be more immature than the CD34þCD38þ and theCD34þCD38 SRCs.
In order to more precisely elucidate the hierarchical organization of human primitive HSCs/hematopoietic progenitor
cells, including CD34 SRCs, CD34þCD38 SRCs, andCD34þCD38þ SRCs, we recently investigated the functional characteristics of these three classes of SRCs [12].
In that study, the CD34 SRCs and CD34þCD38 SRCs
could both support human cell repopulation for up to 24
weeks in NOG mice [12]. On the other hand, theCD34þCD38þSRCs could only support short-term human

Figure 6. Long-term multilineage reconstitution abilities of CD34þ38
and CD34 SRCs. First, the R1 gate was set on the total murine BM cells
obtained from these two representative NOG mice that received five

CD34þCD38 SRCs (left column) or two CD34 SRCs (right column)
24 weeks after transplantation. Thereafter, the human CD45þ cells weregated as R2 (solid line). The expression of surface markers, including
CD34, CD19, CD33, CD14, and CD41, on the R2 gated cells was analyzed
by six-color FCM. Only the expression of CD235a was analyzed using R3gate (dotted line) containing CD45þ/ cells. The percentages of positive
cells in each scattergram (CD45, CD34, CD19, CD33, CD14, andCD41) are presented in the right upper corner. The percentages ofCD235a-positive cells are presented in bilateral upper corners.

M. Ishii et al./ Experimental Hematology 2011;39:203–213

211

Figure 7. T-lymphoid reconstitution abilities of CD34þ38 and CD34 SRCs. The human T cells detected in the BM, PB, and spleen obtained from these
two representative NOG mice that received five CD34þCD38 SRCs (A, upper column) or two CD34 SRCs (B, lower column) 24 weeks after transplantation are presented. First, CD3þ cell gates were set on the human CD45þ cells, and then the expression of CD4/CD8 on the CD3þ cells was analyzed by sixcolor FCM. The percentages of each fraction are indicated.

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cell repopulation in NOG mice as reported previously[12,20]. The frequency of CD34þCD38 SRCs in
13LinCD34þCD38 cells is approximately 1/40 by IBMI
[12]. In contrast, the frequency of CD34 SRCs in
13LinCD34 cells is approximately 1/25,000 by IBMI[12], which is significantly lower than that in CD34þCD38
SRCs. Therefore, it is very important to highly purify the
CB-derived CD34 SRCs in order to more precisely
elucidate their HSC characteristics.
In this study, we added five more Lin-specific mAbs
(anti-CD11b, anti-CD33, anti-CD45RA, anti-CD66c, andanti-CD127) to remove the retained myeloid and lymphoidcells in the 13LinCD34 cell fraction. We then succeededin highly purifying the CD34 SRCs (Fig. 1). An in vivo
limiting dilution analysis demonstrated the frequency of
CD34 SRCs in the 18LinCD34 cells to be approximately 1/1,000 (Fig. 4). The 2  103 18LinCD34 cells(containing only two CD34 SRCs) could support longterm (up to 24 weeks) as well as high-level human hematopoiesis in NOG mice (Fig. 5) and showed extensive
multilineage human cell repopulation into different cell lineages including CD34þ stem/progenitor, CD19þ B-lymphoid,CD4þ/CD8þ T-lymphoid, CD14þ monocytic, CD33þ
myeloid, CD41þ megakaryocytic, and CD235aþ erythroid
lineages (Figs. 6 and 7). These results unveiled that the
CD34 SRCs had potent human cell repopulation potentials
in NOG mice at least for 24 weeks.
In our model, the CD34 SRCs are more immature thanthe CD34þCD38 SRCs, because the CD34 SRCs could
generate CD34þ SRCs in vitro as well as in vivo [9,11].
These results may suggest that the hierarchical position of
CD34þCD38 SRCs is in the intermediate stage of the
HSC compartment between CD34 SRCs andCD34þCD38þ SRCs [13]. However, further studies will
be required to fully elucidate the proposed model of thehuman HSC hierarchy [13]. For this purpose, our goal
was to purify the CD34 SRCs at the single-cell level. In
this study, we developed an efficient purification method
using a negative selection technique to deplete lineagepositive cells. It would be more efficient to use specificpositive markers of the CD34 SRCs/HSCs. Thus, it isinevitably essential to detect/identify novel and/or known
specific positive markers that are expressed on the CD34
SRCs/HSCs. Nakamura et al. recently reported that tie 2,
a receptor tyrosine kinase, is a candidate positive marker
of human BM-derived CD34- SRCs [24]. However, CBderived 18LinCD34 cells did not express the tie 2
receptor (Sasaki and Sonoda, unpublished data). Using
this newly developed high-resolution purification method,
studies to identify specific positive markers of CD34
SRCs are now under investigation in our laboratory.
Acknowledgments
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, Tokyo, Japan; a grantfrom Haiteku Research Center of the Ministry of Education;a grant from the Science Frontier Program of the Ministry of
Education; a grant from the 21st Century Center of Excellence
program of the Ministry of Education; a grant from the Promotionand Mutual Aid Corporation for Private Schools of Japan; a grantfrom Kansai Medical University, Osaka, Japan (Research grant B);
a grant from the Japan Leukemia Research Foundation; a grantfrom the Mitsubishi Pharma Research Foundation; a grant fromthe Takeda Science Foundation, Osaka, Japan; and a grant fromthe Terumo Life Science Foundation, Kanagawa Prefecture,
Japan. All these grants were given to Y. Sonoda.
The authors are grateful to the Keihan Cord Blood Bank forproviding the samples used in this study. Kyowa Kirin Company(Tokyo, Japan) is also acknowledged for providing the various
growth factors used in this study.

Conflict of interest disclosures
No financial interest/relationships with financial interest relating
to the topic of this article have been declared.

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Supplementary Figure E1. RT-PCR analyses for mRNAs of c-kit, Flt3, c-Mpl, and IL-3Ra.

Supplementary Table E1. Primer pairs used for RT-PCR
Gene name
c-kit
Flt-3
c-Mpl
IL-3RaGAPDH

Strand
F
R
F
R
F
R
F
R
F
R

Primer sequences
0

Product length (bp)0

5 -AAGGACTTGAGGTTTATTCCT-3
50 -CTGACGTTCATAATTGAAGTC-30
50 -CCATCTTGGACCTGGAAGAA-30
50 -AAGATGTGCCAAGGGAATTG-30
50 -TGGAGATGCAGTGGCACTTG-30
50 -AGAACTGTGGGGTCTGTAG-30
50 -ACAGGTCAGAGACAGAACCTCC-30
50 - CTGTTCTTCTTCCTGGCAGC-30
50 -ACCACAGTCCATGCCATCAC-30
50 -TCCACCACCCTGTTGCTGTA-30

345
193
206
555
450