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Citation: Blood Cancer Journal (2015) 5, e290; doi:10.1038/bcj.2015.22
www.nature.com/bcj

LETTER TO THE EDITOR

Human cord blood-derived primitive CD34-negative
hematopoietic stem cells (HSCs) are myeloid-biased long-term
repopulating HSCs
Blood Cancer Journal (2015) 5, e290; doi:10.1038/bcj.2015.22;
published online 13 March 2015

Hematopoietic stem cells (HSCs) possess both self-renewal andmulti-lineage differentiation abilities and maintain lifelong hematopoiesis. Recent studies have revealed that the murine HSC
compartment consists of heterogeneous HSC subpopulations interms of their lineage-biased differentiation potentials.1–6 Dykstra
et al. have recently categorized murine HSCs as α-, β-, γ- andδ-cells, according to the contribution ratio of myeloid or lymphoidcells in the repopulation assays.1,2 In addition, other groups havereported that lineage-biased HSCs could be prospectively isolatedby their surface immunophenotypes. For example, CD41+ murine
HSCs have shown to possess a long-term (LT) repopulating
capacity and showed a marked myeloid-biased reconstituting
capacity.3 In addition, the murine platelet-primed von Willebrandfactor (vWF)-positive HSCs have LT-myeloid-biased lineage
repopulation potentials and can self-renew.4 Furthermore, Morita
et al.5 reported that the murine HSC compartment could besegregated according to the expression levels of CD150 antigen,and CD150high HSCs had the most potent self-renewal activities, aswell as the the highest myeloid-biased lineage differentiation
potentials. All of these reports demonstrated that myeloid-biased
murine HSCs have a LT-repopulating capacity and can produce
lymphoid-biased HSCs. Therefore, myeloid-biased HSCs were
thought to be most primitive HSCs in the murine HSC
hierarchy.1–6
Conversely, it has not yet been elucidated whether the human
HSC compartment consists of homogeneous or heterogeneous
HSC subsets in terms of the lineage-biased differentiation
potentials. It has long been believed that human HSCs
and hematopoietic progenitor cells (HPCs) exist only in the
CD34-positive (CD34+) fraction. However, we recently identiﬁed
CD34-negative (CD34−) SCID-repopulating cells (SRCs) in thehuman cord blood (CB) using an intra-bone marrow injection(IBMI) technique.7–13 In addition, we developed high-resolution
puriﬁcation methods for these CB-derived CD34− SRCs using
18Lineage (18Lin)-speciﬁc antibodies.10–13 These highly puriﬁed
lineage-depleted CB-derived CD34− cells possessed not only SRC
abilities but also colony forming cell (CFC) abilities in the
methylcellulose semi-solid culture.10–13 These observations clearly
demonstrated that human CB-derived HSCs/HPCs (HSPCs) exist
not only in the CD34+ but also in the CD34− fraction. However, thefunctional differences between these human CD34+/ − HSPCs are
not fully elucidated. Therefore, in this study, we precisely analyzed
the differences in the differentiation potentials between CD34+/ −HSPCs in vivo and in vitro.
In order to compare the in vivo differentiation potentials of
CD34+/ − SRCs, we ﬁrst performed an SRC assay. The CB-derived
18Lin-negative (18Lin−) CD34+/ − cells, both of which containhighly puriﬁed CD34+/ − SRCs,10 were transplanted into NOD/Shi(NOG) mice using the IBMI technique. Then, thescid/IL-2 Rγnullcpercentages of CD19+, CD33+ and other types of cells (deﬁned as
human CD45+, CD19−, CD33− cells) in the human CD45+ cells

produced from CD34+/ − SRCs in the mouse BMs were seriallyanalyzed (schematically presented in Supplementary Figure S1).
The human CD45+ cell repopulation capacities of both
CD34+/ − SRCs were not signiﬁcantly different (Figure 1a andSupplementary Figure S2). These data were consistent with ourpreviously reported data that both CD34+/ − SRCs possessedcomparable human CD45+ cell repopulation capacities.7–13 However, the differentiation potentials of these CD34+/ − SRCs withregard to the CD33+ myeloid cells were clearly different. These
CD34− SRCs showed signiﬁcantly higher rates of CD33+ myeloid
cell repopulation (Figures 1e and 2). At 5–6, 12 and 18–24 weeks
after transplantation, CD34− SRCs showed signiﬁcantly higher
percentages of CD33+ cells (74.4, 30.4 and 29.8%, respectively;
P o0.01) compared with CD34+ SRCs (22.8, 13.1 and 17.7%,
respectively) in the mouse BMs (Figures 2g–i). Surprisingly, anumber of the mice that received CD34− SRCs showed exclusively
human CD33+ myeloid cell repopulation at 5 weeks after
transplantation (Figure 2d and Supplementary Figure S3B).
However, these CD34− SRCs were not myeloid-committedprogenitors. Because all of the mice received CD34− SRCs
showed multi-lineage human hematopoietic cell reconstitution
at 18–24 weeks after transplantation (Figures 1 and 2,
Supplementary Figures S2 and S3). We have previously reported
that these CD34− SRCs possessed secondary and tertiary (41 year)
multi-lineage reconstituting abilities as did CD34+ SRCs.12 The
percentages of CD33+ cells in the mice that received CD34− SRCs
were gradually decreased from the early-to-late weeks after
transplantations (Figures 2g–i), and concomitantly the percentages of CD19+ B-lymphoid cells increased (Figures 2j–l).
In contrast, CD34+ SRCs produced signiﬁcantly higher percentages of CD19+ cells compared with CD34− SRCs until 12 weeks
after transplantation (Figures 2a, b, j and k). At 5–6, 12 and
18–24 weeks after transplantation, the mean percentages ofCD19+ cells in the mouse receiving both CD34+/ − SRCs were 66.7
and 21.0% (P o 0.01), 80.8 and 64.7% (P o 0.05), and 59.7 and65.6% (P = 0.496), respectively (Figures 2j–l). Therefore, CD34+
SRCs predominantly produced CD19+ cells in the mouse BM at
each time point (Figure 2 and Supplementary Figure S3A). These
results were consistent with recently reported data.14
We next further analyzed the multi-lineage differentiation
potentials of CD34+/ − SRCs. At 18–24 weeks after transplantation, mice were killed and the human hematopoietic multilineage reconstitutions in the mouse left tibia (injection site)were analyzed by FACS. Both CD34+/ − SRCs could produce
comparable levels of CD34+ progenitor cells, CD19+ B lymphocytes, CD14+ monocytes, CD41+ megakaryocytes and CD3+
T lymphocytes in the murine BM (Figures 1b–d, f and g and
Supplementary Figure S2), as we reported previously.10–13
However, CD34− SRCs produced higher percentages of CD33+
cells compared with those of CD34+ SRCs (Figures 1e and 2g–i),
as above-mentioned. On the contrary, CD34+ SRCs produced asigniﬁcantly higher percentage of CD235a+ cells compared with
CD34− SRCs (Figure 1h). Collectively, these results demonstrated,
for the ﬁrst time, that human CB-derived CD34− SRCs are
myeloid-biased SRCs.

Letter to the Editor

2
We further analyzed in vitro the differentiation potentials of
CD34+/ − HSPCs by a CFC assay and coculture with human bonemarrow-derived mesenchymal stromal cell (MSC) feeders, which
support human HSCs, as we recently reported.13 As shown in
Supplementary Figure S4A, in the presence of 30% fetal calf serum(FCS) supplemented with a cocktail of cytokines, including stem
cell factor, interleukin (IL)-3, granulocyte colony-stimulating factor,

CD3

CD45

100

granulocyte/macrophage colony-stimulating factor and erythropoietin (EPO), the plating efﬁciency (PE) of 18Lin−CD34+ cells(69.8%) was signiﬁcantly higher than that of 18Lin-CD34- HSPCs(50.9%) (P o0.05). The 18Lin-CD34+ cells formed all types ofcolonies, including CFU-GM (61.5%), BFU-E (30.2%) and CFU-Mix(7.0%). Conversely, 18Lin−CD34− cells formed mainly BFU-E(57.4%) and CFU-Mix (40.9%) colonies, and only a few myeloid

CD14

80

40

60

30

40

20

% of positive cells

80

n.s.
60

40

n.s.
10

20

20

0

0

CD34+

0

CD34+

CD34-

CD19

CD34-

CD34-

CD34
25

80

80

CD34+

CD33

100

% of positive cells

n.s.

20

n.s.

60

n.s.
60

15
40

p = 0.014

40

10
20
5

20

0

0

CD34+

0

CD34+

CD34-

CD41

25

CD34-

CD34+

CD34-

CD235a16

CD34+ SRC

12

CD34- SRC

% of positive cells

20

15
8

n.s.

10

p = 0.046

4

5

0

0

CD34+

CD34-

CD34+

CD34-

Figure 1. Comparision of the multi-lineage differentiation potentials between CD34+/ − SRCs at 18–24 weeks after transplantation.
At 18–24 weeks after transplantation, the mice were killed and BM cells were collected from the left tibia. (a) The percentages of humanCD45+ cells in the mouse BM with hemolysis. The expression of surface markers including human (b) CD3, (c) CD14, (d) CD19, (e) CD33,(f) CD34 and (g) CD41 on human CD45+ cells are indicated. (h) The percentages of CD235a+ cells on mouse CD45− human CD45+/ − cells in themouse BM without hemolysis. Each open circle represents the data of an individulal mouse. Open and gray bars show the mean percentagesof each marker-positive cell.
Blood Cancer Journal

Letter to the Editor

3

CD33+
5 - 6 wk

18 - 24 wk

60

40

40

40

20

20

20

0

0

0
13
12
14
10
18
11
7
17
15
2
8
5
9
19
3
6
4
16
1

60

14
18
8
15
11
13
10
12
1
3
2
9
4
5
17
7
19
6
16

80

60

Mouse identification number

Mouse identification number

Mouse identification number

5 - 6 wk

12 wk

(%)
100

(%)

11

7

11
8
12
16
4
14
13
17
18
2
15
10
19
1
3
9
6
5
7

0
5

0
6

0

1

20
2

20
9

40

20

3

40

8

60

40

10

80

60

4

80

60

Mouse identification number

Mouse identification number

5 - 6 wk
100

18 - 24 wk

12 wk

p < 0.01

p < 0.01

100

80

80

80

60

60

40

40

40

20

20

20

0

CD34+

0

CD34+

CD34-

5 - 6 wk
100

CD34-

CD34+

12 wk
p < 0.05

100

p < 0.01

100

80

80

80

60

60

60

40

40

40

20

20

20

0

0

CD34+

p < 0.01

100

60

0

18 - 24 wk

100

80

Mouse identification number% of CD33+ cells

(%)

100

80

16
17
19
13
15
18
12
6
14
3
7
10
1
5
2
4
9
11
8

CD34- SRC

12 wk

(%)
100

80

(%)
100

% of CD19+ cells

Others

17
7
18
13
1
12
4
3
11
14
19
2
15
8
6
16
5
10
9

CD34+ SRC

(%)
100

CD19+

CD34-

18 - 24 wk
n.s.

0

CD34+

CD34-

CD34-

CD34+

CD34-

+/ −

Figure 2. Serial analysis of the ratio of CD19 and CD33 cells produced from CD34
SRCs in the mouse BM. The percentages of CD19+,
CD33+ and other type of cells on the human CD45+ cells in the mice BM cells from the right tibia were serially analyzed from 5–24 weeks after
transplantation by the BM aspiration method. The CD19+ or CD33+ cells on the human CD45+ cells were gated as indicated in Supplementary
Figure S2. The human CD45+CD19−CD33− cells were deﬁned as ‘other type of cell’. Each individual mouse was identiﬁed by ear punching andthe human hematopoietic cell repopulation was traced from 5–24 weeks after transplantation. (a–f) The percentages of CD33+ (ﬁlled bar),
CD19+ (open bar) and other types of cells (gray bar) in the human CD45+ cells in the mouse BM from the right tibia. Each bar indicates thedata of an individual mouse receiving (a–c) CD34+ and (d–f) CD34− SRCs. The mean percentages of (g–i) CD33+ and (j–l) CD19+ cells on
human CD45+ cells in the mouse BM at each time point are indicated. The data represent the means ± s.d.
++colonies (1.7%), which is consistent with our recent data.10–13
In addition, in order to further assess the erythroid andmegakaryocyte differentiation potentials of CD34+/ − HSPCs, a
CFC assay was performed in the presence of 10% platelet-poor
plasma supplemented with TPO, EPO and IL-3. Under these
conditions, the PE of 18Lin−CD34− cells (66.9%) was signiﬁcantly
higher than that of 18Lin−CD34+ cells (29.4%) (P o 0.01). The
18Lin−CD34− cells formed erythroid (16.3%), megakaryocyte(29.3%) and erythro-megakaryocytic mixed colonies (54.2%;
Supplementary Figure S4B).

Finally, we analyzed the differentiation potentials of CD34+/ −HSPCs in the coculture system. The fold increase of 18Lin−CD34+
cells (207-fold) was signiﬁcantly greater than that of 18Lin−CD34−
cells (29.9-fold; P o 0.01)), after 7 days cocultuers (Supplementary
Figure S5A). In addition, the number of CD34+ cells maintained/
generated from 1 × 103 18Lin−CD34+ cells (4.6 × 104) was signiﬁcantly higher than that generated from 1 × 103 18Lin−CD34− cells(0.6 x 104) (Po0.01; Supplementary Figure S5B). The numbers of
CD11b+ and CD14+ cells produced from one CD34+ cell generated
from 1 × 103 18Lin−CD34+ cells were signiﬁcantly higher than those
Blood Cancer Journal

Letter to the Editor

4

produced from one CD34+ cell generated from 1 × 103 18Lin−CD34−
cells (Po0.01; Supplementary Figures S5D and E). Conversely, thenumber of CD41+ cells produced from one CD34+ cell generated
from 1 × 103 18Lin−CD34− cells was signiﬁcantly higher than that
produced from one CD34+ cell generated from 1 × 103 18Lin−CD34+
cells (Po0.01; Supplementary Figure S5F). These results are fairly
consistent with the results of the CFC assay, in which 18Lin−CD34−
cells showed poor myeloid colony formation, and mainly formed
erythro-megakaryocytic colonies.
It was recently reported that murine most primitive CD150high
or vWF+ HSCs showed megakaryocyte primed gene expression
patterns, and in the CFC assay, they formed more megakaryocyte
containing colonies compared with CD150int or vWF- HSCs.3–5,15Therefore, it is suggested that human CD34− HSPCs are the humancounterpart of the above-mentioned murine primitive HSCs.
In summary, the present data clearly demonstrated, for the ﬁrst
time, that human CB-derived CD34− SRCs (HSCs) possess myeloidbiased LT-repopulating capacities. We also recently reported that
CD34− SRCs could produce most primitive CD34+ SRCs (Lin−CD34+
CD38−CD90+CD45RA− SRCs) in the cocultures with human bonemarrow cell-derived mesenchymal stromal cells.13 These results
suggest that myeloid-biased CD34− HSCs may produce lymphoidbiased CD34+ HSCs. In other words, the expression of CD34 may
segregate or separate lymphoid-biased HSCs from myeloid-biased
HSCs. Collectively, it may be suggested that the human myeloidbiased LT-repopulating CD34− HSCs reside in the apex of thehuman HSC hierarchy. However, the molecular mechanisms which
control the lineage bias program of the primitive human HSCs
have not yet been determined. Further investigation at the singlecell level is necessary to better elucidate the pathway of human
HSC lineage differentiation.15 These studies also have important
implications for clinical HSC transplantation in patients withmalignant and nonmalignant hematological diseases.
CONFLICT OF INTEREST
The authors declare no conﬂict of interest.

ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid for Scientiﬁc Research C (grant nos.
19591144, 21591251 and 24591432) from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan; a grant from the Science Frontier Programof the MEXT; a grant from the Strategic Research Base Development Program forPrivate Universities from the MEXT; a grant from the Promotion and Mutual Aid
Corporation for Private Schools of Japan; a grant from Kansai Medical University(Research grant B); a grant from the Japan Leukemia Research Foundation; a grantfrom the Mitsubishi Pharma Research Foundation; a grant from the Takeda Science
Foundation; a grant from the Terumo Life Science Foundation; and a grant from the
SENSHIN Medical Research Foundation. The grants from the Takeda Science
Foundation were given to YM and YSo, and all other grants were given to YSo. We
are grateful to the Japanese Red Cross Kinki Cord Blood Bank for providing thesamples used in this study. We also thank the Kyowa Hakko Kirin Company (Tokyo,
Japan) for graciously providing us the various growth factors used in this study.

AUTHOR CONTRIBUTIONS
YM, conception and design of the study, ﬁnancial support, provision of the study
material, collection and/or assembly of the data, data analyzes, statistical analyzes
and interpretation, and writing the manuscript; KS, HK, RN, TF and YSa, provision of
the study material, collection and/or assembly of the data; YSo, conception anddesign of the study, ﬁnancial support, administrative support, data analyzes andinterpretation, writing of the manuscript and ﬁnal approval of the manuscript.

Y Matsuoka1, K Sumide1, H Kawamura1,2, R Nakatsuka1, T Fujioka1,
Y Sasaki1 and Y Sonoda1
1
Department of Stem Cell Biology and Regenerative Medicine,
Graduate School of Medical Science, Kansai Medical University,
Hirakata, Osaka, Japan and2
Department of Orthopedic Surgery, Kansai Medical University,
Hirakata, Osaka, Japan
E-mail: sonoda@hirakata.kmu.ac.jp
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