J. Biomedical Science and Engineering, 2013, 6, 53-58 JBiSE
doi:10.4236/jbise.2013.68A1005 Published Online August 2013 (http://www.scirp.org/journal/jbise/)
In vitro differentiation of human umbilical cord-derived
mesenchymal stem cells into CD34+ cells via CD34
antibody
Shengnan Guo1,2, Libin Guo1, Maoxiang Sun1,2, Wenyue Ma1,2, Yujia Lu1,2, Ying Liu1*
1Department of Stem Cell Clinical Application Central, Siping Hospital Affiliated to China Medical University, Siping, China
2Tuhua Bioengineering Company Ltd., Siping, China
Received 10 June 2013; revised 15 July 2013; accepted 30 July 2013
Copyright © 2013 Shengnan Guo et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
CD34+ cells differentiated from mesenchymal stem
cells (MSCs) have a strong biological function in car-
diovascular regeneration. However, the molecular me-
chanisms of and the methods to improve the CD34+
cell differentiation from MSCs, especially from hu-
man MSCs (hUC-MSCs) are still unclear. In the cur-
rent study, the effect of CD34 antibody on the CD34+
cell differentiation from human umbilical cord (UC)-
derived MSCs (hUC-MSCs) is determined. The re-
sults have demonstrated that the expression of cd34
protein is significantly increased in hUC-MSCs t reate d
with CD34 antibody. In addition, the cell prolifera-
tion is increased in hUC-MSCs after treatment with
CD34 antibody. Moreover, the expression of PI3K,
AKT, p-AKT proteins, which are signaling molecules
related to stem cell differentiation, is increased by
CD34 antibody. The results suggest that CD34 anti-
body could promote the differentiation of hUC-MSCs
into CD34+ cells and PI3K/AKT may be involved in
this important process.
Keywords: Differentiation; Human Umbilical
Cord-Derived Mesenchymal Stem Cells; CD34+ Cells
1. INTRODUCTION
CD34+ mesenchymal stem cell (MSC) transplantation
has received growing attention as a promising treatment
for cardiovascular diseases [1,2]. MSCs, being a hetero-
geneous stromal cell population, having the capacity to
self-renew as well as to differentiate into a variety of ter-
minally differentiated cells, including adipocytes, chondro-
cytes, endothelial cells, and osteocytes [3-5]. MSCs also
have the capacity to home in the sites of vascular injury,
to engraft into damaged blood vessels, to differentiate into
vascular cells, and to exert a paracrine effect by the local
release of vascular growth factors and cytokines [4,6,7].
However, the molecular mechanisms of and the methods
to improve the CD34+ cell differentiation from MSCs,
especially from human MSCs (hUC-MSCs) are still un-
clear.
CD34 antibody has been widely used as a molecular
tool to separate a sub-group of MSCs (CD34+-MSCs) for
stem cell studies and for stem cell therapeutic applica-
tions. However, the potential biological functions of CD34
antibody in CD34 expression and CD34+ cell differentia-
tion of MSCs are currently unknown. In this study, we
are trying to determine the effect of the CD34 antibody
on the expression of CD34 in cultured human umbilical
cord (UC)-derived MSCs (hUC-MSCs) and the potential
mechanisms involved.
2. MATERIALS AND METHODS
2.1. Harvesting of UC
Five human UC samples were collected from the mothers,
in accordance with the ethical committee of Institute of
Siping Central Hospital. Informed consent was obtained
from all subjects. All studies and laboratory procedures
were carried out in the Siping Central Hospital. The sec-
tions of umbilical cords were internally washed with phos-
phatebuffered saline (PBS) containing 300 U/ml penicil-
lin and 300 ug/ml streptomycin (Gibco, Grand Island, NY),
and immediately immersed in Dulbecco’s modified Ea-
gle’s medium-low glucose (DMEM-LG; Gibco) supple-
mented with 10% fetal bovine serum (FBS; Gibco), 300
U/ml penicillin, and 300 ug/ml streptomycin. All sam-
ples were processed within 12 - 15 hours after collection.
*Corresponding author.
Published Online August 2013 in SciRes. http://www.scirp.org/journal/jbise

S. N. Guo et al. / J. Biomedical Science and Engineering 6 (2013) 53-58 54
2.2. Isolation and Culture of Adherent Cells
from UC
As described in our previous study [8], UCs were filled
with 0.1% collagenase (Sigma-Aldrich, St. Louis) in PBS
and incubated at 37˚C for 20 min. Each UC was washed
with proliferation medium, and the detached cells were
harvested after gentle massage of the UC. Cells were
centrifuged at 300 g for 10 min, resuspended in prolif-
eration medium, and seeded in 75-cm2 flasks at a density
of 5 × 107 cells per ml. After 24 h of incubation, non-
adherent cells were removed, and culture medium was
added with CD34 antibody (0.1 ug/ml) and replaced eve-
ry 2 days. Adherent cells were cultured until they reach-
ed 80% - 90% confluence.
2.3. Flow Cytometry
The hematopoietic lineage marker CD45, human leuco-
cyte antigen HLA-DR, the cell-surface markers of endo-
thelial progenitor cells CD31 and CD90, were deter-
mined by flow cytometry. In this experiment, the adher-
ent cells were incubated for 2 h at 37˚C with the rabbit
antihuman fluorophore-conjugated primary antibodies
(all diluted 1:1000 in 0.01 M PBS; all from Becton Dick-
inson, Franklin Lakes, NJ, USA) for direct immunofluo-
rescence staining. A total of 10,000 labeled cells were
analyzed using a Becton Dickinson LSRFortessaTM Cell
Analyzer with the Becton Dickinson FACSDivaTM soft-
ware version 6.0 (Becton Dickinson Biosciences, San
Jose, CA, USA).
2.4. Cell Proliferation Assay
hUC-MSCs were incubated with CD34 antibody (0.01
μmol/l, 0.1 μmol/l or 1 μmol/l) for 96 h. The effects of
CD34 antibody on proliferation were assessed by cell
counting. hUC-MSCs without CD34 antibody treatment
were selected as a control group. Cell counts were ex-
pressed as a percentage of the control.
2.5. Immunofluorescence Staining.
hUC-MSCs were incubated with CD34 antibody (0.01
μmol/l, 0.1 μmol/l or 1 μmol/l, defined as A, B and C
group) for 7 d or 14 d. hUC-MSCs without CD34 antibody
treatment were used as a control group. Adherent cells
were fixed with 4% cold paraformaldehyde (Sigma) for
15 min and permeabilized with 0.1% Triton X-100 (Sigma)
for 10 min. After washing with PBS, cells were blocked
with 3% bovine serum albumin (BSA, Sigma) for 30 min,
followed by incubating overnight at 4˚C with mouse
anti-human primary antibody against CD34 (1:200, Ab-
cam), or rabbit anti-human primary antibody against
CD105 (1:200, Abcam). The FITC-labeled secondary an-
tibody (1:100; goat polyclonal to rabbit IgG; Abcam) and
PRITC-labeled secondary antibody (1:100; goat poly-
clonal to mouse IgG; Abcam) were then added and in-
cubated for 1 h at 37˚C temperature. Cells were washed
three times with PBS and counter-stained with DAPI
(4’,6-diamidino-2-phenylindole) nuclear dye. The im-
munofluorescence staining was observed using Leica
DM5000 B fluorescence microscope.
2.6. Western Blot Analysis
hUC-MSCs were incubated with CD34 antibody (0.01
μmol/l, 0.1 μmol/l or 1 μmol/l) for 7 d or 14 d. hUC-
MSCs without CD34 antibody treatment were used as a
control group. Homogenates were prepared from these
MSCs using radioimmunoprecipitation assay buffer as
described previously [8]. Equal amounts of protein (20 -
40 μg) were added and separated on a 14% Tris-glycine
gel, transferred to a polyvinylidene difluoride membrane,
and incubated with a PI3K, AKT or a p-AKT primary
antibody (Amyjet Scientific Inc, Wuhan, China) at a di-
lution of 1:500. The membranes were incubated with an
anti-rabbit horseradish peroxidase antibody and develop-
ed by enhanced chemiluminescence. The blots were then
treated with a protein stripping buffer, reblocked with a
blocking agent, and then immunostained with an anti-
body against human β-actin (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) as the loading controls.
2.7. Statistical Analysis
All data are expressed as mean ± SEM (standard error of
the mean). All the experiments were repeated independ-
ently at least 3 times. Data were analyzed using analysis
of variance followed by Student’s t test when only two
groups were studied, taking p < 0.05 as significant.
3. RESULTS
3.1. Isolation and Culture of Adherent Cells
from UC
Similar to our previous studies [8], primary adherent cul-
tures were generated from all UCs samples, with cells
displaying a mesenchymal stem cell (MSC)-like phenol-
type. After 4 days in culture the cells formed colonies,
reaching confluence after 10 - 14 days. Most of the cells
were spindle-shaped, resembling fibroblasts. After the
second passage, adherent cells constituted homogeneous
cell layers with an MSC-like phenotype. The number of
adherent cells from the UCs decreased slightly after freez-
ing and thawing, and the remaining viable cells were
successfully expanded on consecutive days (data not
shown).
3.2. Immunophenotype
Very few of the adherent cells derived from the UC sam-

S. N. Guo et al. / J. Biomedical Science and Engineering 6 (2013) 53-58 55
ples had haematopoietic lineage markers CD45, endothe-
lial markers (CD31) and HLA-DR (HLA-class II) on the
cell surface, as assessed by flow cytometry. The majority
of the cells presented high levels of CD90 protein on the
cell surface. In comparison with the fibroblast control, no
obvious difference in the levels of surface antigens could
be observed, which were similar to our previous studies
[8].
3.3. Cell Proliferation Assay
The effect of CD34 antibody on cell proliferation was as-
sessed by cell counting. Compare with the control group,
hUC-MSCs incubated with CD34 antibody (0.01 μmol/l,
0.1 μmol/l or 1 μmol/l) for 96 h proliferated at 21%, 53%,
28% separately (Table 1).
3.4. Immunohistological Findings
To further study the biological characteristics of the dif-
ferentiated CD34+ cells from hUC-MSCs, we determined
the expression of CD34 and CD105 on the cells’ surface
at 7, 14 days after treatment with CD34 antibody (Figure
1). CD34 expression was identified by red fluorescence,
whereas CD105 expression was displayed by green fluo-
rescence. Many green cells were observed in every time
point. The numbers of red cells in these slices were sig-
nificantly higher (P < 0.05) than green cells. In addition,
as shown in Figures 1(b) and (c), and F-H, we observed
small, solitary yellow fluorescence which indicated that
cells treated with CD34 antibody could co-express CD34
and CD105 protein. More cells express CD34 protein
(red fluorescence) than CD105 protein (green fluores-
cence) in C group at 14 d, which suggested the loss of
CD105 protein.
3.5. Expression of AKT Protein
PI3K, AKT, p-AKT proteins were expressed in the diffe-
rentiation process of hUC-MSCs incubated with CD34
antibody (0.01 μmol/l, 0.1 μmol/l or 1 μmol/l) for 7 d
and 14 d. Compare with the control group, hUC-MSCs
were incubated with CD34 antibody (0.01 μmol/l, 0.1
μmol/l or 1 μmol/l) for 7 d, PI3K protein increased by
7%, 23%, 43% separately; AKT and p-AKT were similar
Table 1. hUC-MSCs were incubated with CD34 antibody (0.01
μmol/l, 0.1 μmol/l or 1 μmol/l) for 96 h. The effects of CD34
antibody on proliferation were assessed by cell counting. hUC-
MSCs without CD34 antibody treatment were selected as a
control group. Cell counts were expressed as a percentage of
the control. Compared with the control group, hUC-MSCs were
incubated with CD34 antibody (0.1 μmol/l or 1 μmol/l) for 96 h
proliferated 48.6%, 28.3% separately. *P < 0.05.
Control 0.01 μmol/l 0.1 μmol/l 1 μmol/l
0.45 ± 0.02 0.42 ± 0.01 0.83 ± 0.03* 0.59 ± 0.02
to PI3K protein express (see Figures 2(a) and (b)). At 14
d, the expression of PI3K, AKT, p-AKT proteins was
higher than that at 7 d (see Figures 2(a) and (b)).
4. DISCUSSION
In the present study, we have demonstrated for the first
time that CD34 antibody could promote hUC-MSCs to
co-express CD34 and CD105 proteins. While agreeing
with the previously report [9,10], during this process, we
have observed the cultured cells express CD105 (green
staining in Figure 1) at the early phase, and express
CD34 (red staining in Figure 1) in the following days.
MSCs are a heterogeneous stromal cell population, and
cells display some phenotypic and functional characteris-
tics of endothelial cells. In our previous study, changes in
hUC-MSCs have been detected utilizing multiple cul-
ture methods and we have observed the osteodifferention
of hUC-MSCs [8]. Changes in hUC-MSCs are coupled
with human maladies [11,12]. However, these cells re-
quire laborious and time intensive culture techniques.
Until now, the differentiation of hUC-MSCs remains un-
defined, in part because there is not a consensus defini-
tion by cell surface antigen expression, such as CD34
and CD105. CD34 has been used as biomarkers in endo-
thelial progenitor cells (EPCs) as well as angiogenesis
therapies [13,14]. CD105 and CD34 have been found at
the sites of blood vessel formation [15,16]. In this study,
the expression of CD34 and CD105 in different phase sug-
gests that CD34 antibody could promote the hUC-MSC
differentiation into CD34+ cells.
Multiple signaling pathways are involved in the dif-
ferentiation of MSCs. Akt is known to be part of a pro-
survival pathway and the phosphorylation at Thr308 is
required for Akt activation [17,18]. In the current study,
we have found that PI 3-kinase and phospho-Akt (Thr308)
are elevated in CD34 antibody-treated hUC-MSCs com-
pared with the control group. However, the effect of
CD34 antibody-mediated up-regulation of CD34 expres-
sion is reduced when the concentration of the CD34 anti-
body reaches at 1 umol/l. The result suggests that maxi-
mal effect of CD34 antibody on the CD34 expression
may occur at 0.1 umol/l. These observations were com-
plied with survival of hUC-MSCs in 96 h at different
CD34 antibody concentrations. It is still unclear why Akt
(Thr308) is changed, since this would promote but not
inhibit apoptosis. However, other phosphorylation sites
or downstream effectors may be involved in the prosur-
vival and differentiated effect of hUC-MSCs-condition-
ed media.
Limitations of this current study were: 1) the lack of
cell proliferation assay continuous data, which could
have confirmed a direct positive effect of CD34 antibody
on hUC-MSC differentiation capability; 2) The lack of

S. N. Guo et al. / J. Biomedical Science and Engineering 6 (2013) 53-58 56
(a)
(b)
Figure 1. CD34 and CD105 double positive cells immunofluorescence findings. (a) Immunofluorescence Findings (CD34 protein:
red fluorescence; CD105 protein: green fluorescence). A. CD105 protein expression of hUC-MSCs incubated without CD34 antibody
at 7 d; B. CD105 protein expression of hUC-MSCs incubated with 0.01 μmol/l CD34 antibody at 7 d; C. CD105 and CD34 protein
expression of hUC-MSCs incubated with 0.1 μmol/l CD34 antibody at 7 d; D. CD105 and CD34 protein expression of hUC-MSCs
incubated with 1 μmol/l CD34 antibody at 7 d; E. CD105 protein expression of hUC-MSCs incubated without CD34 antibody at 14d;
F. CD105 and CD34 protein expression of hUC-MSCs incubated with 0.01 μmol/l CD34 antibody at 14 d; G. CD105 and CD34 pro-
tein expression of hUC-MSCs incubated with 0.1 μmol/l CD34 antibody at 14 d; H. CD105 and CD34 protein expression of
hUC-MSCs incubated with 1 μmol/l CD34 antibody at 14 d; (b) The percentage of CD34 and CD105 double positive cells analysis.
The data were shown as the percentage of CD34 and CD105 double positive cells of the total cells.
telomerase activity analysis, which could have confirmed
maturation of cells; 3) The lack of the necessary func-
tional assays to verify these cells function in vessel for-
mation in vivo. Further controlled basically experiments
will be required to confirm our preliminary findings and
to elucidate the full characterization and mechanistic de-
termination involved in improving hUC-MSCs differen-
tiation, following treated with CD34 antibody.
In summary, our work demonstrated a new method to
promote hUC-MSC differentiation via CD34 antibody.
Administration of CD34 antibody is able to accelerate
the differentiation of hUC-MSCs into CD34+ cells and
AKT may play a role in this process. These findings re-
inforce our previous observation on the importance of
JBiSE

S. N. Guo et al. / J. Biomedical Science and Engineering 6 (2013) 53-58 57
(a1) (a2)
(b1) (b2)
Figure 2. Expression of protein. (a) Expression of protein. PI3K, AKT, p-AKT protein expressed in the differentiation phase of
hUC-MSCs incubated with CD34 antibody (0.01 μmol/l, 0.1 μmol/l or 1 μmol/l) as A, B and C group for 7 d and 14 d separately; con:
control group. (a1) Expression of protein in 7 d; (a2) Expression of protein in 14 d. (b) Expression of protein analysis. (b1) Ex-
pression of protein analysis in 7 d. Compared with the control group, hUC-MSCs incubated with CD34 antibody (0.01 μmol/l, 0.1
μmol/l or 1 μmol/l) for 7 d, PI3K protein expression increased 7%, 23%, 43% separately; AKT and p-AKT protein expression were
similar to PI3K protein express; (b2) Expression of protein analysis in 14 d. Compared with the control group, hUC-MSCs incubated
with CD34 antibody (0.01 μmol/l, 0.1 μmol/l or 1 μmol/l) for 14 d, PI3K protein expression increased 24%, 46% in A, and B groups
separately; Compared with the B group, PI3K protein expression decreased 26% in C group. *p < 0.05; **p < 0.01; #p < 0.05.
hUC-MSCs for research and future therapeutic use.
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