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Leukemia (2013), 1–8& 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13
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ORIGINAL ARTICLE

CD133 is a positive marker for a distinct class of primitive human
cord blood-derived CD34-negative hematopoietic stem cells
M Takahashi1,2, Y Matsuoka1, K Sumide1, R Nakatsuka1, T Fujioka1, H Kohno1, Y Sasaki1, K Matsui3, H Asano4, K Kaneko2 and Y Sonoda1
The identiﬁcation of human CD34-negative (CD34  ) hematopoietic stem cells (HSCs) provides a new concept for the hierarchyin the human HSC compartment. Previous studies demonstrated that CD34  severe combined immunodeﬁciency (SCID)repopulating cells (SRCs) are a distinct class of primitive HSCs in comparison to the well-characterized CD34 þ CD38  SRCs.
However, the puriﬁcation level of rare CD34  SRCs in 18 lineage marker-negative (Lin  ) CD34  cells (1/1000) is still very low
compared with that of CD34 þ CD38  SRCs (1/40). As in the mouse, it will be necessary to identify useful positive markers for a highdegree of puriﬁcation of rare human CD34  SRCs. Using 18Lin  CD34  cells, we analyzed the expression of candidate positivemarkers by ﬂow cytometric analysis. We ﬁnally identiﬁed CD133 as a reliable positive marker of human CB-derived CD34  SRCs andsucceeded in highly purifying primitive human CD34  HSCs. The limiting dilution analysis demonstrated that the incidence ofCD34  SRCs in 18Lin  CD34  CD133 þ cells was 1/142, which is the highest level of puriﬁcation of these unique CD34  HSCs todate. Furthermore, CD133 expression clearly segregated the SRC activities of 18Lin  CD34  cells, as well as 18Lin  CD34 þ cells,in their positive fractions, indicating its functional signiﬁcance as a common cell surface maker to isolate effectively both CD34 þ
and CD34  SRCs.
Leukemia advance online publication, 26 November 2013; doi:10.1038/leu.2013.326
Keywords: CD133; hematopoietic stem cell; CD34 negative; SCID-repopulating cell; IBMI; cord blood

INTRODUCTION
The sialomucin, CD34, has long been believed to be a major
positive marker for murine and human hematopoietic stem cells(HSCs) and hematopoietic progenitor cells (HPCs).1–4 Despite
the importance of the CD34 antigen as a marker of early HSCs/
HPCs in basic research, as well as in clinical HSC transplantation,
the biological function of CD34 has not been fully elucidated.1
During the past two decades, the ﬂuorescence-activated cellsorting (FACS) technology has markedly improved, and it is now
possible to isolate prospectively murine HSCs using various cellsurface markers, including Sca-1, c-kit and CD150.2–6 In fact, thesurface immunophenotype of murine HSCs has been increasingly
reﬁned, resulting in the success of the single cell-basedtransplantation analyses.7–9 Using such advanced technology,
Nakauchi and his co-workers revealed that murine long-term
lymphohematopoietic-reconstituting HSCs are lineage markernegative (Lin  ) c-kit þ Sca-1 þ CD34-low/negative (CD34lo/ KSL).10 Collectively, these results established the concept thatthe most primitive murine HSCs are present in the CD34lo/  KSL
cell population.
In contrast, the current understanding of the primitive human
HSC compartment is far behind that of the murine HSC system.
As in the mouse, the puriﬁcation of primitive human HSCs requires
positive markers. It is well documented that CD34 is expressed on
the surfaces of HSCs, as well as the more differentiated HPCs.1–4
Therefore, it is important to identify additional positive markers forthe puriﬁcation of primitive human HSCs at the single-cell level.
The severe combined immunodeﬁciency (SCID)-repopulating cell(SRC) xenotransplantation assay provides a gold standard
surrogate assay for human HSCs.11 A number of studies using
xenograft assays demonstrated that the bone marrow (BM)- andcord blood (CB)-derived primitive human HSCs are enriched in the
Lin  CD34 þ CD38  population.2–4,11–14 It was also reported that
the Lin  CD34 þ CD38  CD90 þ CD45RA  CB fraction contains
primitive HSCs, and this activity has been isolated to as
few as 10 puriﬁed cells.15 Recently, Notta et al.16 published
an informative study showing that CB-derived single
Lin  CD34 þ CD38  CD45RA  Thy1 þ RholoCD49f þ cells were highly
efﬁcient in generating long-term multilineage engraftment in
NOD-SCID-IL-2Rgc  /  mice,16 providing strong evidence thatprimitive human CD34 þ CD38  HSCs express CD49f.
Although the most primitive murine HSCs are deﬁned as
CD34  KSL cells,10 primitive human HSCs were believed to beLin  CD34 þ CD38  cells.2–4,11–16 However, this long-standing
dogma was challenged by several groups.17–20 Interestingly,
these studies suggested that CD34 þ HSCs originate from
CD34  HSCs. Accordingly, we developed an intra-BM injection(IBMI) method and successfully identiﬁed human CB-derived
CD34  SRCs using 13 lineage-speciﬁc monoclonal antibodies(mAbs) to remove Lin þ cells.21,22 These CD34  SRCs could
generate CD34 þ CD38  SRCs in vitro as well as in vivo.21–23
Moreover, our in vivo kinetic analysis clearly demonstrated that
only one or two CD34  SRCs had potent human hematopoietic
cell reconstitution potentials, which was almost equivalent to thatof 5–10 CD34 þ CD38  SRCs.24,25 Using this highly efﬁcient SRC
assay, we demonstrated that the surface immunophenotype

1
Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Hirakata, Osaka, Japan; 2Department of Pediatrics,
Kansai Medical University, Hirakata, Osaka, Japan; 3Department of Gynecology and Obstetrics, Fukuda Hospital, Kumamoto, Japan and 4School of Nursing, Kyoto Prefectural
University of Medicine, Kyoto, Japan. Correspondence: Professor Y Sonoda, Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science,
2-5-1 Shinmachi, Kansai Medical University, Hirakata 573-1010, Osaka, Japan.
E-mail: sonoda@hirakata.kmu.ac.jp
Received 8 October 2013; accepted 17 October 2013; accepted article preview online 5 November 2013

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al

MATERIALS AND METHODS
Collection of CB samples and processing of Lin  cells
CB samples were obtained from normal full-term deliveries with signed
informed consent. This study was approved by the Institutional Review
Board of Kansai Medical University. The CB-derived Lin  mononuclear cells
were separated using an EasySep Human Progenitor Cell Enrichment Kit(StemCell Technologies, Vancouver, BC, Canada) and manipulated by using
the RoboSep (StemCell Technologies) according to the manufacturer’s
instructions.

Immunostaining of Lin  cells and puriﬁcation of Lin  CD34 þ / 
CD133 þ /  cells
The pooled Lin  cells from multiple donors were stained with various
mAbs (see Supplementary Table S1) for 30 min on ice in Ca2 þ  andMg2 þ -free phosphate-buffered saline (PBS  ) (Nakalai Tesque, Kyoto,
Japan) containing 2% fetal calf serum (FCS) (Bioﬁll, Elsternwick, VIC,
Australia) (PBS  /FCS). We used ﬂuorescein isothiocyanate-conjugated
18Lin mAbs against CD2, CD16, CD24 and CD235a (Dako, Kyoto, Japan);
CD3, CD7, CD10, CD11b, CD20, CD41 and CD66c (Beckman Coulter,
Fullerton, CA, USA); CD19 and CD56 (BD Biosciences, San Jose, CA, USA);
CD4, CD14, CD33 and CD127 (eBioscience, San Diego, CA, USA); CD45RA(Southern Biotech, Birmingham, AL, USA); a Paciﬁc Blue-conjugatedanti-CD45 mAb (BioLegend, San Diego, CA, USA); an apophycocyanin(APC)-conjugated anti-CD34 mAb (BD Biosciences) and a phycoerythrin(PE)-conjugated anti-CD133 mAb (Miltenyi Biotec, Bergish Gladbach,
Germany). The cells were then washed once thoroughly with PBS  /FCS,
and resuspended in a 7-amino-actinomycin D (7-AAD) (Beckman Coulter)containing PBS  /FCS solution before the ﬂow cytometric (FCM) analyses
or FACS. For all multicolor analyses, Fluorescence Minus One controls,
wherein all other speciﬁc mAbs were present in the same tube minus theone of the interest, which is replaced with an appropriate isotype-matched
control, were included for determining the ﬂuorescent threshold. The
stained cells were then sorted into four fractions, including 18Lin  CD34 þ / 
CD133 þ /  cells, using a FACS Aria III (BD Biosciences) (Figure 1).

Clonal cell culture
Colony-forming cells (CFCs) were assayed using our standard methylcellulose cultures as reported.21,23,25

Coculture with human BM-derived mesenchymal stromal cells andthe analysis of culture-generated cells by FCM
First, we established human BM-derived mesenchymal stromal cells (MSCs)
using Lin  CD45  cells as reported.28 In this study, we used the MSCs
established from human BM-derived Lin  CD45  CD271 þ SSEA-4 þ cells
designated as DP MSCs, which expressed a high level of the nestin geneand had supporting potential for human CB-derived CD34 þ /  SRCs.
A total of 1  103 puriﬁed 18Lin  CD34 þ /  CD133 þ /  cells per wellof a 24-well plate (BD Falcon, San Jose, CA, USA) were plated onto
Leukemia (2013) 1 – 8

a

d
CD133
R3
83.6%

R4
0.3%

b
R2
54.9%

R5
75.0%

R6
3.4%

CD45

e
CD45

CD133

FSC

c
CD34

R1
81.5%

7AAD

of CD34  SRCs is Lin  CD34  c-kit  ﬂt3  .23,26 However, thefrequency of CD34  SRCs in these 13Lin  CD34  cells isapproximately 1/25 000, which is still very low in comparison to
the frequency (1/40) of CD34 þ CD38  SRCs in 13Lin  CD34 
cells.21,22,24 To more effectively purify CD34  SRCs, we thendeveloped a high-resolution puriﬁcation method using 18Lin
mAbs, which can enrich CD34  SRCs to 1/1000.25
As our goal is to purify the CD34  HSCs to the single-cell level,
it was necessary to identify speciﬁc positive markers for CD34 
HSCs. Using these 18Lin  CD34  cell populations, we extensively
analyzed the expression of candidate positive markers, including
known HSC markers and various adhesion molecules, by FACS.
Finally, we identiﬁed CD133, a ﬁve-transmembrane glycoprotein,27
as a positive marker of human CB-derived CD34  SRCs (HSCs) andsucceeded in highly purifying primitive human CD34  SRCs
(HSCs) to the level of 1/142 cells. Moreover, CD133 expression
clearly segregated the SRC activities of 18Lin  CD34  cells, as well
as 18Lin  CD34 þ cells, in their positive fractions. These results
indicate that CD133 is a common cell surface maker that can beused to isolate effectively both CD34 þ and CD34  SRCs.

SSC

2

R7
13.5%

R8
46.5%

18Lin

CD45

Figure 1. Representative FACS profiles of CB-derived 18Lin  CD45 þ
CD34 þ /  CD133 þ /  cells. (a) The 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 18Lin  living
cells. (c) The 18Lin  living cells (R2) were subdivided into two
fractions: 18Lin  CD45 þ CD34 þ (R3) and CD34  (R4) cells, according to their expression levels of CD34. The definitions of CD34 þ / 
cells are as follows: the CD34 þ fraction contains cells expressing
maximum APC fluorescence intensity (FI) to 5% the level of FI.
The CD34  level of FI was determined based on the Fluorescence
Minus One controls. (d) The 18Lin  CD34 þ cells residing in the R3gate were further subdivided into two fractions: 18Lin  CD45 þ
CD133 þ (R5) and CD133  (R6) cells, according to their expression
levels of CD133. The definitions of CD133 þ /  cells are as follows:
the CD133 þ fraction contains cells expressing maximum PE FI to
15% the level of FI and the CD133  level of FI was determinedbased on the Fluorescence Minus One controls. (e) The R4-gatedcells were further subdivided into two fractions: 18Lin  CD45 þ
CD34  CD133 þ (R7) and CD133  (R8) cells. The definitions ofCD133 þ /  cells are the same as described above.
pre-established irradiated (1200 cGy using a 137Cs-g irradiator) DP MSC layers
in StemPro-34 medium (Gibco Laboratories, Grand Island, NY, USA); a cocktail
of cytokines including 50 ng/ml stem cell factor (ATgen, Gyeonggi, South
Korea), 50 ng/ml ﬂt3 ligand (FL) (R&D Systems, Minneapolis, MN, USA),
100 ng/ml thrombopoietin, 10 ng/ml interleukin (IL)-3 (R&D Systems),
100 U/ml intereukin-6 (IL-6) (kindly provided by Ajinomoto Co. Inc., Tokyo,
Japan) and 10 ng/ml granulocyte colony-stimulating factor and 5% FCS(HyClone Laboratories, Logan, UT, USA). The granulocyte colony-stimulating
factor and thrombopoietin 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 the cell counting using acounting chamber. The cells were then stained with 7-AAD, an APCconjugated anti-CD34 mAb (BD Biosciences) and a Paciﬁc Blue-conjugatedanti-CD45 mAb (BioLegend). The number of CD34 þ cells and theexpression of various lineage markers, including CD33, CD14, CD11b andCD41, was analyzed by ﬁve-color FCM (FACS CantoII; BD Biosciences), as
described below.

NOG mice
Six-week-old female NOD/Shi-SCID/IL-2Rgnull(NOG) mice29 were purchasedcfrom the Central Institute of Experimental Animals (Kawasaki, Japan). All
mice were handled under sterile conditions and were maintained in germfree 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.21–25

The SRC assay and the serial analysis of human CD45 þ cellengraftment in NOG mice by FCM
The SRC assays were performed using a previously reported method.21–25
In this study, 5  103 puriﬁed CB-derived 18Lin  CD34 þ /  CD133 þ /  cells& 2013 Macmillan Publishers Limited

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al3were transplanted by IBMI into sublethally irradiated (250 cGy using a
137
Cs-g irradiator) 8-week-old NOG mice. The repopulation of humanCD45 þ hematopoietic cells in the NOG mouse BM was serially analyzed by
our aspiration method, as reported.21,24,25 In brief, 1–2 ml of BM ﬂuids wereserially aspirated from the contralateral sites (right tibia) of each mouse 6,
12 and 18 weeks after transplantation, and the BM ﬂuid was then diluted
with PBS  /FCS plus 1% heparin. The mice were ﬁnally killed 18–20 weeks
after transplantation and the BM cells were collected by crushing the pairsof femurs and tibiae of each mouse in a mortar. The cells were suspended
in PBS  /FCS and the remaining bone tissues were removed using a cellstrainer (BD Falcon). The repopulation of human hematopoietic cells in themurine BM was determined by detecting the number of 7-AAD  cells
positively stained with Paciﬁc Blue-conjugated anti-human CD45 mAb bysix-color FCM (FACS CantoII; BD Biosciences). The cells were also stainedwith a PE-Cy7-conjugated anti-mouse CD45.1 mAb (Beckman Coulter);
ﬂuorescein isothiocyanate-conjugated anti-human CD19 (eBioscience),
CD11b (Beckman Coulter) and CD235a (DAKO) mAbs; PE-conjugatedanti-human CD33, CD14 and CD41 (Beckman Coulter); and an APCconjugated anti-human CD34 mAb (BD Biosciences) for the detection ofhuman stem/progenitor, B-lymphoid and myeloid hematopoietic cells.
For the precise analysis of the T/NK-cell development in the thymus andspleen, the cells obtained from these organs were stained with ﬂuorescein
isothiocyanate-conjugated anti-human CD3 (Beckman Coulter), PE-conjugated anti-human CD4 and an APC-conjugated anti-human CD8 mAbs
(eBioscience) and a ﬂuorescein isothiocyanate-conjugated anti-humanCD56 mAb (BD Biosciences).
The mice were scored as positive if more than 0.01% of the total murine
BM cells were human CD45 þ cells. In separate experiments, we conﬁrmed
the detection limit of human CD45 þ cells in mouse BMs was 0.005%(Supplementary Figure S1).

Secondary transplantation
For secondary transplantations, murine BM cells were obtained 18–20
weeks after transplantation from the pairs of femurs and tibiae ofengrafted primary recipient NOG mice that received 5  103 18Lin 
CD34 þ /  CD133 þ cells. One out of the ﬁve portions of the whole BM cells
were transplanted by IBMI into sublethally (250 cGy) irradiated secondary
recipient NOG mice. Eighteen weeks after transplantation, the presence ofhuman CD45 þ cells in the secondary recipients’ BM was analyzed by FCM,
as described for primary transplantation.

In vivo limiting dilution analysis
To assess the frequency of SRCs in the CB-derived 18Lin  CD34  CD133 þcells, various numbers of cells (200, 400 and 800; n ¼ 15) were transplanted
into NOG mice by IBMI, as reported.21–26 The mice were killed 12 weeks
after transplantation, and the human cell repopulation in the mouse BM
was analyzed by FCM as described above.

Statistical analyses
In the limiting dilution analysis (LDA) (Figure 6), the frequencies of SRCs
were calculated using a software application for the LDA as reported.30
In Figures 2 and 3 and Supplementary Figure S3, the differences in themean colony numbers or expression levels of each pair were examined by
two-tailed Student’s t-test.

RESULTS
Identiﬁcation of candidate positive markers of CD34  SRCs (HSCs)
First, we extensively explored candidate positive markers that
could be useful for the efﬁcient puriﬁcation of rare CB-derived
CD34  SRCs (HSCs), including known HSC markers and various
adhesion molecules, using highly puriﬁed CB-derived 18Lin 
CD34 þ /  cells25 by multicolor FCM. Representative data areshown in Supplementary Figure S2. Known HSC markers, including
Tie2, KDR, ABCG2 and CD150, were not expressed on either
18Lin  CD34 þ or 18Lin  CD34  cells. On the other hand, CD133,
c-kit, CD90, CXCR4, CD49f and CD93 were expressed on both typesof cells. The 18Lin  CD34 þ /  cells could be subdivided into
positive and negative fractions. In order to select a good positivemarker from these candidate molecules, we back-gated thepositive fractions of these markers in 18Lin  CD34  cells into
& 2013 Macmillan Publishers Limited

forward scatter/side scatter (FSC/SSC) scattergrams. As clearlyseen in Supplementary Figure S2, the distribution of c-kit þ ,CD90 þ , CXCR4 þ , CD49f þ and CD93 þ cells in the 18Lin  CD34 
cells was scattered inside and around the blast window.
In contrast, the 18Lin  CD34  CD133 þ cells were concentrated
in the low FSC blast window. On the basis of these data,
we selected CD133 as a candidate positive marker for humanCB-derived CD34  SRCs (HSCs).
Puriﬁcation of human cord blood-derived 18Lin  CD34 þ / 
CD133 þ /  cells
First, the R1 gate was set on the blast–lymphocyte window(Figure 1a). Next, the 7-AAD  18Lin  cells were gated as R2,
as shown in Figure 1b. The 18Lin  CD45 þ CD34 þ and 18Lin 
CD45 þ CD34  cells were then gated as R3 and R4 (Figure 1c),
respectively. The 18Lin  CD45 þ CD34 þ /  cells were further
subdivided into four distinct populations gated as R5 (18Lin 
CD45 þ CD34 þ CD133 þ ), R6 (18Lin  CD45 þ CD34 þ CD133  ),R7 (18Lin  CD45 þ CD34  CD133 þ ) and R8 (18Lin  CD45 þ CD34 
CD133  ), based on their surface CD133 expression (Figures 1dand e). Then, the 18Lin  CD34 þ CD133 þ /  (R5 and R6) and
18Lin  CD34  CD133 þ /  (R7 and R8) fractions were sorted forthe subsequent in vitro cultures and in vivo SRC assays. As shownin Figures 1d and e, 75% of the 18Lin  CD45 þ CD34 þ fraction (R3)
and 13.5% of the 18Lin  CD45 þ CD34  fraction (R4) expressed
CD133.
The hematopoietic colony-forming capacity of CB-derived
18Lin  CD34 þ /  CD133 þ /  cells
The CFC capacities of the CB-derived 18Lin  CD34 þ and CD34  cells
were unique (Figure 2). The plating efﬁciencies of 18Lin  CD34 þ
CD133 þ , CD34 þ CD133  , CD34  CD133 þ and CD34  CD133 cells were 57%, 65%, 39% and 19%, respectively. Interestingly,
the 18Lin  CD34  CD133 þ /  cells mainly formed erythroid burst(BFU-E) (71 and 73%) and colony-forming unit (CFU)-Mix(mostly erythro/megakaryocytes containing mixed colonies)
colonies (25 and 27%). On the contrary, they formed few
CFU-granulocyte/macrophage (CFU-GM) colonies (4 and 0%).
On the other hand, the 18Lin  CD34 þ CD133 þ cells formed alltypes of CFCs, including CFU-GM, BFU-E and CFU-Mix. It is ofinterest that the 18Lin  CD34 þ CD133  cells formed mainly BFUE (68%) and CFU-Mix (30%) colonies, and few CFU-GM (2%)
colonies, which was a CFC pattern similar to that of the 18Lin 
CD34  cells. These results are consistent with our previous
report,25 and again conﬁrmed that the CFC capacities can bedetected in the CB-derived 18Lin  CD45 þ CD34  cell fraction.
These results indicated that the CB-derived 18Lin  CD45 þ CD34 cell fraction contains HPCs.
Coculture with human BM-derived MSCs (DP MSCs)
As reported,21,23 the CD34  SRCs could produce CD34 þ SRCsin vitro. Therefore, 1  103 18Lin  CD34 þ /  CD133 þ /  cells
were cocultured with human BM-derived MSCs (DP MSCs),28
in the presence of six cytokines for 1 week. The 18Lin 
CD34 þ /  CD133 þ /  cells actively proliferated and maintained/
generated CD34 þ cells (Figures 3a–c). In the cocultures of 18Lin 
CD34 þ CD133 þ /  cells, the total number of cells expanded by
130- (1.3  105 cells) to 450- (4.5  105 cells) fold, which contained
approximately 30% of the CD34 þ cells, resulting in a signiﬁcantly
higher number of CD34 þ cell recovery in the coculture with
CD34 þ CD133 þ cells. On the other hand, the total number ofcells derived from 18Lin  CD34  CD133 þ /  cells expanded by
30- (3  104 cells) to 130- (1.3  105 cells) fold. Both the 18Lin 
CD34  CD133 þ /  cells produced CD34 þ cells. However, thepercentage and absolute number of CD34 þ cells produced from
18Lin  CD34  CD133 þ cells (31.7% and 3.2  104 cells) weresigniﬁcantly higher than those of 18Lin  CD34  CD133  cells
Leukemia (2013) 1 – 8

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al

4
CD34+

CD34-

*

a

(x 105)
6

CFU-mix

Total No. of output cells/well

BFU-E
CFU-GM

**

100

50

CD133+

CD133-

CD133+

CD133-

Figure 2. Colony-forming capacities of sorted 18Lin  CD45 þ
CD34 þ /  CD133 þ /  cells. Two hundred sorted 18Lin  CD45 þ
CD34 þ /  CD133 þ /  cells were cultured in methylcellulose at
37 1C with 5% CO2, 5% O2 and 90% N2 in the presence of stem
cell factor, thrombopoietin, IL-3, granulocyte/macrophage colonystimulating factor, granulocyte colony-stimulating factor and erythropoietin for 12 to 14 days. The number and composition ofvarious colonies derived from the 18Lin  CD45 þ CD34 þ /  CD133 þ / 
cells are shown. The types of colonies identified in situ were
granulocytes (CFU-G), macrophages (CFU-M), granulocyte/macrophage (CFU-GM), erythroid burst (BFU-E) and erythrocyte-containing
mixed (CFU-Mix). Open, shaded and closed bars represent the
number of CFU-Mix, BFU-E and CFU-GM (including CFU-G, CFU-M
and CFU-GM) colonies, respectively. The numbers of all the types ofhematopoietic colonies were determined as the mean of quadruple
cultures. The data represent the means±s.d. *Po0.05; **Po0.01.

(13.2% and 0.4  104 cells). In addition, both the 18Lin  CD34 
CD133 þ /  cells generated higher percentages (13.5 and 11.5%)of CD41 þ cells compared with those produced from the 18Lin 
CD34 þ CD133 þ /  (1.8 and 4.2%) cells (Supplementary Figures
S3–D). In contrast, the 18Lin  CD34 þ /  CD133 þ cells generated
signiﬁcantly higher percentages of CD33 þ , CD11b þ and CD14 þcells than did the 18Lin  CD34 þ /  CD133  cells (Supplementary
Figure S3A–C). Collectively, these results indicated that the 18Lin 
CD34  CD133 þ /  cell fraction contained a substantial numberof CD34 þ cell-producing HPCs/HSCs, and that the 18Lin 
CD34 þ /  CD133 þ /  cells showed different in vitro lineage
differentiation potential.

b

Leukemia (2013) 1 – 8**

2CD133+

CD133-

CD133+

CD34+

CD133-

CD34-

n.s.
**

25

0

(x 105)
2

CD133+

CD133-

CD133+

CD34+

CD133-

CD34-

**

1
**

0

SRC activities and multilineage differentiation potentials of
CB-derived 18Lin  CD34 þ /  CD133 þ /  cells following IBMI
Thereafter, we investigated the SRC activity of the 18Lin  CD34 þ / 
CD133 þ /  cells using NOG mice. Previously, Lapidot and his coworkers reported elegant studies showing that the chemokine
stromal cell-derived factor-1 (CXCL12) and its receptor, CXCR4, hasa pivotal role in the homing and repopulation of CD34 þ SRCs in
NOD/SCID mice.31,32 Moreover, it was recently reported that thestromal cell-derived factor-1–CXCR4 axis modulates homing, BM
retention and the mobilization of HSPCs in a more complex way
than was thought previously.33 We previously demonstrated that
CB-derived CD34  SRCs expressed lower levels of homing
receptors, including CXCR4, and that they could not migrate
toward the gradient of stromal cell-derived factor-1 in transwell
cultures.21
These data suggested that CB-derived CD34  SRCs cannot
home into the BM niche following conventional tail vein injection.
We previously demonstrated that human CB-derived CD34  SRCs
could only be detected following the IBMI method.21–26 On thebasis of these results, 5  103 18Lin  CD34 þ /  CD133 þ /  cells
were transplanted by IBMI into NOG mice, and the human cell
repopulation was analyzed 18–20 weeks after transplantation.
Interestingly, all 10 mice that received 18Lin  CD34 þ /  CD133 þ

CD34-

4

(%)
50

c

CD34+
**

0

% of CD34+ cells/well0

Absolute No. of CD34+
cells/well

No. of colonies/200 cells

150

CD133+

CD133

þ

CD133+
þ/

CD133þ/

Figure 3. Coculture of 18Lin CD45 CD34
CD133
cells withDP MSCs. A total of 1  103 purified 18Lin  CD45 þ CD34 þ CD133 þ / 
cells (left panel) and 18Lin  CD45 þ CD34  CD133 þ /  cells (rightpanel) were cocultured with DP MSCs in the presence of six
cytokines (SCF þ FL þTPO þ IL-3 þ IL-6 þ G-CSF) for 1 week. Eachcoculture contained 22 wells. (a) The total numbers of output cells
per well. (b) The percentages of CD34 expression on the culturegenerated CD45 þ cells are shown. (c) The absolute numbers of
CD34 þ cells maintained/produced in cocultures with DP MSCs areshown. The data represent the means±s.d. **Po0.01; NS, notsignificant; G-CSF, granulocyte colony-stimulating factor; SCF, stem
cell factor; TPO, thrombopoietin.

cells (ﬁve mice each) were repopulated with human CD45 þ cells(Table 1). The level of human cell engraftment was 63.1 to 88.0%(mean, 73.9%) for 18Lin  CD34 þ CD133 þ cells and 11.0 to 52.7%(mean, 30.4%) for 18Lin  CD34  CD133 þ cells. In contrast, noneof the 12 mice that received 18Lin  CD34 þ /  CD133  cells (sixmice each) were repopulated with human cells. These results
demonstrated, for the ﬁrst time, that the CD133 is a positivemarker for human CB-derived CD34  SRCs, and also that CD133
expression clearly segregates the SRC activities of the 18Lin 
CD34 þ /  cells in their positive fraction.
& 2013 Macmillan Publishers Limited

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al5the thymus in both types of NOG mice. The CD56 þ natural killer
cells were also detected in the spleens in both types of NOG mice.
These results conﬁrmed that both the CD34 þ /  CD133 þ SRCs had
multilineage differentiation potentials.

To further evaluate the functional differences between the
CD34  CD133 þ and CD34 þ CD133 þ SRCs, we studied theirmultilineage reconstitution abilities in various organs, including
the BM, spleen and thymus, in NOG mice that were transplantedwith 5  103 of 18Lin  CD34 þ /  CD133 þ cells by IBMI. The resultsof the repopulation patterns are shown in Figure 4. The analysesof the two representative mice that were transplanted with either
CD34 þ CD133 þ SRCs (Figure 4a, upper column) or CD34 
CD133 þ SRCs (Figure 4b, lower column) demonstrated both of
the SRCs to have a comparable in vivo differentiation capacity into
CD34 þ stem/progenitor cells, B-lymphoid, myeloid, monocytic,
megakaryocytic and erythroid lineages at 18–20 weeks after thetransplantation. In addition, CD4 þ and CD8 þ single- and doublepositive T-lymphoid cells were detected in the CD3 þ cell gates ofLong-term repopulation patterns of CD34 þ /  CD133 þ SRCs in
NOG mice and their secondary repopulating abilities
The next approach to characterize the self-renewal potential andthe long-term repopulating potential of the CD34  SRCs was toserially analyze the kinetics of BM engraftment for 18 weeks in
NOG mice that received transplants of 5  103 18Lin  CD34 þ / 
CD133 þ cells (Figures 5a and b).
In these experiments, both the mice that received transplants of
CD34 þ /  CD133 þ SRCs showed signs of human cell repopulation
at 6 weeks after the transplantation. The repopulation level of all
ﬁve mice that received both types of SRCs gradually increased,
and reached the peak levels at 12–18 weeks after transplantations.
As we mentioned above, none of the mice that received 5  103
18Lin  CD34 þ /  CD133  cells were repopulated with humancells (Figures 5a and b).
We then performed the secondary transplantation to analyze
their self-renewing capacities of the cells. All of the secondary
recipient mice that received the transplants from the primary
recipient mice that received 5  103 of either of the 18Lin 
CD34 þ /  CD133 þ cells by IBMI showed signs of human cell
repopulation 18 weeks after the transplantation (Supplementary
Table S2). The human CD45 þ cell rates in all the secondary
recipient mice showed comparable levels of human cell repopulation (0.01–0.03% vs 0.04–0.14%). We conﬁrmed that all of the

63.1–88.0 (73.9)0 (0)11.0–52.7 (30.4)
0 (0)

5/5
0/6
5/5
0/6

Abbreviations: BM, bone marrow; CB, cord blood; SRC, SCID-repopulatingcell. aThe human cell repopulation in the right tibia of primary recipient
mice was analyzed 18 weeks after transplantation by BM aspiration.

BM

hCD45

50.5

R3

43.8

hCD45

hCD45

CD4

hCD45

BM

BM

BM

BM

BM

43

CD19

CD33
hCD45

hCD45

BM

BM

36.5

hCD45

70.5

0.21

0.02

8.08

hCD45

CD14

hCD45

CD34

7AAD
CD11b

mCD45.1

FSC

R2

41.2

5.06

2.64

19.6

98.4

SSC

SSC

92.6

Spleen12.3

CD41

CD34-CD133+

hCD45

Thymus64.2

hCD45

Spleen

44.9

19.3

20.6

hCD45

Thymus
CD8

b

R4

0.02

5.36

hCD45

hCD45

BM
0.65

BM

3.84

R3

hCD45

R1

12.2

CD235aR4

CD11b

mCD45.1

BM

7AAD
75.8

BM

75.1

hCD45
CD235a

FSC

BM

25.5

CD19

CD34

97.9
R2

SSC

SSC

97.3
R1

BM

12.7

6.8

47.4

6.79

CD56

CD34+CD133+

CD33

a

CD14

5000
5000
5000
5000

18Lin  CD34 þ CD133 þ
18Lin  CD34 þ CD133 
18Lin  CD34  CD133 þ
18Lin  CD34  CD133 

CD56

Incidence ofengraftment

CD8% of humanCD45 þ cellsa(mean)

CD3

No. oftransplanted
cells

CD3

Type of cells

CD41

The SRC activity of sorted CB-derived 18Lin  CD34 þ / 
CD133 þ /  cells

Table 1.

0.79

hCD45

hCD45

CD4

hCD45

hCD45

Figure 4. Long-term multilineage reconstitution abilities of CD34 þ CD133 þ and CD34  CD133 þ SRCs. First, the R1 gate was set on the total
murine BM cells obtained from these two representative NOG mice that received (a) CD34 þ CD133 þ SRCs (upper column) and (b) CD34 
CD133 þ SRCs (lower column), 20 weeks after transplantation. Thereafter, the human CD45 þ cells were gated as R3 (solid line) from the R2gated 7-AAD  cells. The expression of surface markers, including CD34, CD33, CD19, CD41, CD14 and CD11b, on the R3-gated cells wasanalyzed by six-color FCM. Only the expression of CD235a was analyzed using the R4 gate (dotted line) containing CD45 þ /  cells. The
percentages of positive cells in each scattergram (CD45, CD34, CD33, CD19, CD41, CD14, CD11b and CD235a) are presented in the indicatedsquares. The expression of CD3, CD4 and CD8 on the human CD45 þ cells in the thymus, and the expression of CD56 on the human CD45 þ
cells in the spleen was also analyzed and presented. The percentages of positive/negative cells in each scattergram are indicated.
& 2013 Macmillan Publishers Limited

Leukemia (2013) 1 – 8

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al618Lin-CD34-CD133+
No. of cells transplanted

(%)
100
0

75

% Human CD45+ cells in the mouse BM

400

600

800

37
f=1/31350

CD34+CD133+
CD34+CD133-

25

10

f=1/14210
f=1/65

0

b

200

100

% negative mice

% Human CD45+ cells in the mouse BMa6
12
Weeks After transplantation

18
0.1

50

Figure 6. The frequency of SRCs in the 18Lin  CD34  CD133 þ cells.
Various numbers of 18Lin  CD34  CD133 þ cells (200, 400 and 800)
were transplanted to NOG mice (n ¼ 15). The human CD45 þ cell
repopulation in the mouse BM was analyzed by FCM 12 weeks after
transplantation. The frequency of SRCs was one per 142 18Lin 
CD34  CD133 þ cells. For the frequency determination, the middle
solid line represents the estimated weighted mean frequency (f WM).
The lower and upper dotted lines represent the 95% confidence
interval of the fWM.

25

present method can enrich the CD34  SRCs 180 times more thanthe previously described negative selection method.21(%)
100
CD34-CD133+
CD34-CD13375

0
0

6
12
Weeks after transplantation

18

Figure 5. Long-term human hematopoietic cell reconstitution in
NOG mice. A total of 5  103 18Lin  CD34 þ /  CD133 þ /  cells(a and b) was injected into the left tibiae of NOG mice by IBMI. The
human CD45 þ cell rates in the contralateral sites of recipient mice
were serially analyzed by the BM aspiration method 6, 12 and 18
weeks after transplantation by six-color FCM. The data represent themeans±s.d. of the results from five or six mice at each time point.

secondary recipient mice that received transplants of CD34 
CD133 þ , as well as CD34 þ CD133 þ SRCs, showed multilineage
human hematopoiesis, including CD19 þ and CD33 þ cells(Supplementary Figure S4).
These results demonstrated that both the CD34  CD133 þ andCD34 þ CD133 þ SRCs had comparable secondary reconstituting
abilities, and could sustain long-term (up to 38 weeks) humanhematopoiesis in NOG mice, suggesting that both types of SRCs
had signiﬁcant self-renewing potentials.
LDA of CB-derived 18Lin  CD34  CD133 þ cells by IBMI
We ﬁrst reported that the frequency of the CB-derived CD34 
SRCs in the 13Lin  CD34  cells was 1/25 000.21 Then, wedeveloped a high-resolution puriﬁcation method improving ournegative selection method, resulting in an incidence of CD34 
SRCs of 1/1000.25 In this study, we succeeded in more highly
purifying the CD34  SRCs using the identiﬁed positive marker,
CD133. As expected, a LDA using the IBMI demonstrated thefrequency of the CD34  SRCs in the 18Lin  CD34  CD133 þ cells
to be 1/142, which is the highest level of puriﬁcation of thisunique CD34  HSC population to date. (Figure 6). Therefore, theLeukemia (2013) 1 – 8

DISCUSSION
Until recently, all human HSCs/HPCs were believed to be present
in the Lin  CD34 þ cell fraction.1–4 However, the existence ofthe human CD34  HSCs was suggested by several studies.17–20
Bhatia et al.17 ﬁrst reported that SRCs were present in human BMand CB-derived Lin  CD34  cells using tail vein injection.
However, the incidence of CD34  SRCs in the Lin  CD34  cell
population was 1/125 000, which was very low. In that report, they
demonstrated that the frequency of CD34  SRCs increased to
1/38 000 cells after 4 days of short-term culture of these Lin  CD34 
cells in the presence of a cocktail of cytokines. This group later
reported that ex vivo culture of the AC133 þ subset from humanCB-derived CD34  CD38  Lin  cells allowed for a detection of
SRC activity in NOD/SCID mice by a Southern blot analysis.34
However, the levels of human chimerism in the BM of recipient
mice were very low, even after ex vivo culture. Collectively, theseresults suggested that homing receptors, including CXCR4, may
become upregulated on CD34  SRCs during the culture, resulting
in the improvement of their engraftment potential by tail vein
injection. The existence of long-term repopulating CD34  HSCs inhuman CB- and BM-derived Lin  cells was also supported by data
showing that the CD34  fraction of normal human BM contains
cells capable of engraftment and differentiation into CD34 þprogenitors, as well as multiple lymphohematopoietic lineages,
using the human/sheep competitive engraft model.18 However,studies on human CD34  HSCs have been hindered by the lack ofa positive marker comparable to Sca-1 in mice. In this study, we
tried to identify useful/reliable positive markers for highly
purifying rare CD34  HSCs.
We recently developed a high-resolution puriﬁcation method
using 18Lin mAbs (the previously reported 13Lin mAbs21 plus ﬁve
Lin mAbs against CD11b, CD33, CD66c, CD45RA and CD127),
which can enrich CD34  SRCs to 1/1000.25 Using 18Lin  CD34 
cells as a target population, we extensively analyzed theexpression of candidate positive markers, including known HSC& 2013 Macmillan Publishers Limited

CD133 is a positive marker of CD34-negative HSC
M Takahashi et al7markers and various adhesion molecules, by multicolor FCM.
We ﬁnally identiﬁed CD133 as a reliable positive marker for humanCB-derived CD34  SRCs (HSCs), and succeeded in highly purifying
primitive human CD34  HSCs at an incidence of 1/142 cells. This
puriﬁcation level of human CD34  SRCs (HSCs) is the highest
reported in the literatures to date. The present data are consistent
with earlier studies demonstrating that the AC133 þ CD34 þ
population from the CB and BM is enriched for SRC activity.35,36
It is very important to clarify the molecular mechanisms
controlling the maintenance, quiescence, proliferation and differentiation of very primitive CD34  HSCs. For this purpose, it will benecessary to highly purify these CD34  HSCs. This is because if acrude Lin  CD34  cell population is used, then these cells may
contain many contaminating lymphocytes and myeloid cells, as
shown in Figure 1b and Supplementary Figure S5. Therefore, wecannot obtain accurate information about the gene expression
and signal transduction that control the above-mentioned
processes in very primitive CD34  HSCs. To truly elucidate themolecular mechanisms controlling the maintenance, quiescence,
proliferation and differentiation of very primitive CD34  HSCs, itwill be necessary to better purify CD34  HSCs.
Very recently, Bonnet and co-workers37 reported that
CD93 is a positive marker of human CB-derived very
primitive CD34  HSCs. They transplanted Lin  CD34  CD93 þ orLin  CD34  CD38  CD93 þ cells, which did not express CD133, by
tail vein injection, and demonstrated their SRC activity. In contrast,
our identiﬁed CB-derived CD34  SRCs could only be detected by
IBMI.21–26 Furthermore, as shown in Supplementary Figure S2,
CD93 was only marginally expressed on our highly puriﬁed
18Lin  CD34  cells. When we back-gated these cells into the FSC/
SSC scattergram, the 18Lin  CD34  CD93 þ cells were scatteredinside and around the blast window. We further analyzed theexpression of CD93 on the CB-derived 18Lin  CD34  CD133 þcells, which contained all of the CD34  SRC activity in the CB
demonstrated by the present study. As clearly seen in
Supplementary Figure S5, we did not detect any CD93 þ cells in
our 18Lin  CD34  CD38  /loCD133 þ cell population. As reported,
their Lin  CD34  CD38  CD93 þ cells did not express CD133.
However, almost all of the Lin  CD34  CD38  CD93 þ cells were
included in the 18Lin þ cell fraction (Supplementary Figures
S5–R27). This is not surprising because they only depleted mature
cells from their target cells by immunomagnetic beads using the
StemSep (StemCell Technologies), which only removed strongly
Lin þ cells. These immunomagnetically Lin-depleted cells stillcontained many lymphocytes, monocytes, granulocytes andnatural killer cells (see Figure 1b and Supplementary Figure S5),
which expressed CD93.38 Human CB-derived CD45RA þ -naive
T-lymphocytes were previously reported to express CD93.39
Indeed,their
incidenceof
CD34 
SRCs
in
the



þ
Lin CD34 CD38 CD93 cells was 1/B6100 cells, which isvery low compared with our present data (1/142). A comparison of
the results of these two studies is summarized in Supplementary
Table S3. Overall, their data are exactly the opposite of our data,thus suggesting the possibility that there may be different classesof CD34  HSCs in the primitive human HSC compartment. Further
studies will therefore be required to clarify this important issue.
As our goal is to purify the CD34  HSCs at the single-cell level,
it is inevitable that additional speciﬁc positive markers for CD34 
HSCs will need to be identiﬁed besides CD133. Using our newly
developed high-resolution puriﬁcation method, studies to identify
additional speciﬁc positive markers of CD34  HSCs are now
underway in our laboratory. In our model, the CD34  SRCs weremore immature than the CD34 þ CD38  SRCs, as the CD34  SRCs
could generate CD34 þ SRCs in vitro as well as in vivo,21,23 and thelimited number of CD34  SRCs transplanted into primary
recipient mice sustained primary, secondary and tertiarytransplantation (over 1 year) in NOG mice, as we recently
reported.40 These results may suggest that the hierarchical& 2013 Macmillan Publishers Limited

position of CD34 þ CD38  SRCs is in the intermediate stage ofthe HSC compartment, between CD34  SRCs and CD34 þ CD38 þ
SRCs.24,26 However, further studies will be required to fully
elucidate the proposed model of the human HSC hierarchy.24,26
In summary, the present results clearly demonstrated that
CD133 is a reliable positive marker of human CB-derived CD34 
SRCs (HSCs). Furthermore, CD133 expression clearly segregatedthe SRC activities of 18Lin  CD34  cells, as well as 18Lin  CD34 þ
cells, in their positive fractions, indicating that CD133 is a common
cell surface marker for both CD34 þ /  SRCs. More importantly,
these ﬁndings suggest that the number of CD133 þ cells in cord
blood units is a more appropriate marker to detect/predict the
HSC potential of cord blood stem cell transplantation incomparison to the currently used CD34 þ cell numbers.
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.
21591251 and 24591432) from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan; a grant from the Science Frontier Program of the MEXT;
a grant from the Strategic Research Base Development Program for Private
Universities from the MEXT; the MEXT-Supported Program for the Strategic Research
Foundation at Private Universities; a grant from the Promotion and Mutual Aid
Corporation for Private Schools of Japan; a grant from the Japan Leukemia Research
Foundation; a grant from the Mitsubishi Pharma Research Foundation; a grant fromthe Takeda Science Foundation; a grant from the Terumo Life Science Foundation
and a grant from SENSHIN Medical Research Foundation. We are grateful to theJapanese Red Cross Kinki Cord Blood Bank for providing the samples used in thisstudy. Kyowa Kirin Company (Tokyo, Japan) is also acknowledged for providing thevarious growth factors used in this study.

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Leukemia (2013) 1 – 8& 2013 Macmillan Publishers Limited