Cotransplantation of human umbilical cord-derived mesenchymalstem cells and umbilical cord blood-derived CD34+ cellsin a rabbit model of myocardial infarction

Tong Li • Qunxing Ma • Meng Ning •

Yue Zhao • Yuelong Hou

Received: 29 July 2013 / Accepted: 18 October 2013 / Published online: 29 October 2013

� Springer Science+Business Media New York 2013

Abstract The objective of the study is to investigate the

effect of hypoxic preconditioning on the immunomodulatory

properties of human umbilical cord-derived mesenchymal

stem cells (hUC-MSCs) and the effect of cotransplantation

of hUC-MSCs and human umbilical cord blood (hUCB)-

derived CD34? cells in a rabbit model of myocardial

infarction. hUC-MSCs with or without hypoxic precondi-

tioning by cobalt chloride were plated in a 24-well plate, and

then cocultured with hUCB-CD34? cells and PBMCs for

96 h at 37 �C in a 5 % CO2 incubator. For the negative

control, hUC-MSCs were omitted. The groups were divided

as follows: A1 = HP-MSCs ? hUCB-CD34? cells ?

PBMC, A2 = hUC-MSCs ? hUCB-CD34? cells ? PBMC,

Negative Control = hUCB-CD34? cells ? PBMC. Culture

supernatants of each group were collected, and the IL-10 and

IFN-c levels were measured by ELISA. A rabbit model of MI

was established using a modified Fujita method. The animals

were then randomized into three groups and received intra-

myocardial injections of 0.4 ml of PBS alone (n = 8, PBS

group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or

hUC-MSCs ? CD34? cells in PBS (n = 8, Cotrans group),

at four points in the infarct border zone. Echocardiography

was performed at baseline, 4 weeks after MI induction, and

4 weeks after cell transplantation, respectively. Stem cell

differentiation and neovascularization in the infracted area

were characterized for the presence of cardiac Troponin I

(cTnI) and CD31 by immunohistochemical staining, and the

extent of myocardial fibrosis was evaluated by hematoxylin

and eosin (H&E) and Masson’s trichrome. IFN-c was

27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and

IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ±

1.80 pg/ml for the Control, A1 and A2 groups, respectively,

which indicated that hUCB-CD34? cells induced immune

reaction of peripheral blood mononuclear cells, whereas

both hUC-MSCs and HP-MSCs showed an immunosup-

pressive effect, which, however, was attenuated by hypoxic

preconditioning. The Cotrans group had less collagen

deposition in the infarcted myocardium and better heart

function than the hUC-MSCs or PBS group. The presence of

cTnI-positive cells and CD31-positive tubular structures

indicated the differentiation of stem cells into cardiomyo-

cytes and neovascularization. The microvessel density was

12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ±

2.45/HP for the Cotrans group, respectively (P \ 0.01). As a

conclusion, both hUC-MSCs and HP-MSCs have an

immunosuppressive effect on lymphocytes, which, however,

can be attenuated by hypoxic preconditioning. Cotrans-

plantation of hUC-MSCs and hUCB-CD34? cells can

improve heart function and decrease collagen deposition in

post-MI rabbits. Thus, a combined regimen of hUC-MSCs

and hUCB-CD34? cells would be more desirable than either

cells administered alone. This is most likely due to the

increase of cardiomyocytes and enhanced angiogenesis in

the infarcted myocardium.

Keywords Myocardial Infarction � Umbilical cord

mesenchymal stem cells � Umbilical cord blood-

derived CD34? Cells � Cotransplantation

Li Tong and Ma Qunxing have contributed equally to this study.

T. Li (&) � Q. Ma � M. Ning � Y. Zhao � Y. Hou

Tianjin Third Central Hospital, Tianjin, China

e-mail: litong3zx@sina.com

M. Ning

e-mail: lemon2104@sina.com

Q. Ma

The Third Central Clinical College of Tianjin Medical

University, Tianjin, China

123

Mol Cell Biochem (2014) 387:91–100

DOI 10.1007/s11010-013-1874-5

Introduction

Acute myocardial infarction (AMI) promotes an irrevers-

ible and massive loss of cardiomyocytes, followed by

gradual replacement of these damaged cardiomyocytes

with fibrous non-contractile cells and eventually heart

failure [1, 2]. Cellular cardiomyoplasty holds great promise

for the repair or regeneration of infarcted myocardium, in

which exogenous stem cells, such as umbilical cord-

derived mesenchymal stem cells (UC-MSCs) [3, 4] and

peripheral blood/umbilical cord blood (PB/UCB)-derived

CD34? cells [5, 6], are injected into the damaged

myocardium.

Bone marrow (BM) represents the most widely used

source of allogeneic MSCs, it is, however, limited by the

availability of donors because BM aspiration is painful and

may pose risks and complications to some donors.

Umbilical cord matrix or Wharton’s jelly has been sug-

gested as an alternative source of MSCs for the repair and

regeneration of the infarcted or ischemic cardiovascular

tissues [4, 7]. The frequency of hematopoietic stem cells

and progenitor cells in UCB equals or even exceeds that of

BM, and human umbilical cord blood (hUCB) contains up

to tenfold higher amounts of CD34? endothelial precursor

cells as non-mobilized adult peripheral blood [8, 9]. Sev-

eral animal studies have shown that CD34? cells could

differentiate into vascular endothelial cells that contribute

to the increase in the number of microvessels and

improvement of heart function [6, 10].

MSCs are known to improve heart function via angiogen-

esis induced by pro-angiogenic factors, the effect of which can

be increased by hypoxic preconditioning [11]. Zhou et al. [12]

and Weiss et al. [13] have also shown that UC-MSCs have low

immunogenicity and immunomodulatory properties. A

question arises whether these immunomodulatory properties

are retained in hypoxic preconditioned UC-MSCs, which will

be addressed in this study.

Despite their therapeutic potential and advantages

compared with BM-MSCs, there have been few studies on

the use of UC-MSCs and UCB-CD34? cells [14], and to

our knowledge no studies about the cotransplantation of

UC-MSCs and UCB-CD34? for the treatment of MI. In

line with previous findings, it is hypothesized in this study

that cotransplantation of UC-MSCs and UCB-CD34?

might have a better effect than either cells administered

alone in post-MI animals.

Materials and methods

The study protocol was approved by the Institutional

Review Board of Tianjin Medical University and the

Human Research Ethics Committee of Tianjin Third

Central Hospital. All participants provided written

informed consent, and all animals received humane care in

compliance with the Guide for the Care and Use of Lab-

oratory Animals.

Isolation and culture of human UC-MSCs

Human umbilical cords were collected from consenting

mothers in the maternity ward of our hospital. They were

exhaustively washed with PBS to remove residual blood

clots and blood vessels, minced into small pieces of

approximately 1–2 mm3 in size, and then incubated with

0.1 % type IV collagenase (GIBCO, USA) for 60 min.

After centrifugation and washing with PBS, the tissues

were resuspended in low-glucose DMEM/F12 (Bioroc,

Tianjin, China) supplemented with 10 % fetal bovine

serum (FBS, GIBCO, USA) and 100,000 U/ml of penicil-

lin/streptomycin, and then cultured in a humidified 5 %

CO2 incubator at 37 �C.

Isolation and culture of peripheral blood mononuclear

cells (PBMCs)

Human PBMCs were isolated from the peripheral blood of

health donors by Ficoll Histopaque (1.077 g/ml) density

gradient centrifugation (MD Pacific, Tianjin, China), and

the cell concentration was adjusted to 1 9 106/ml with

RPMI 1640 medium (GIBCO, USA).

Isolation and culture of hUCB-CD34? cells

hUCB was also obtained from the mothers. Red cells were

removed by sedimentation in 6 % hydroxyethyl starch (HES,

Fresenius Kabi, Germany), and then the mononuclear cells

were isolated from hUCB by a density gradient centrifugation

method, from which the CD34 cells were positively selected

by immunomagnetic bead separation using a human CD34

Microbead kit (Miltenyi Biotec, Germany). The selected

CD34? cells were plated in a T-25 culture flask in the

STEMPRO�-34 SFM complete medium (GIBCO, USA).

Flow cytometry

Human umbilical cord-derived mesenchymal stem cells (hUC-

MSCs) (2 9 105) in the third passage (P3) were trypsinized,

suspended in 200 ll PBS, and then incubated for 30 min at

room temperature with PE- or FITC-conjugated mouse anti-

human monoclonal antibodies (CD34, CD45, CD90, and

CD105). Mouse isotype antibodies served as controls. The

resuspended cells were washed and then subjected to flow

cytometry (FACSort, B-D Co., USA). The purity of the iso-

lated CD34? cells was also detected by flow cytometry.

92 Mol Cell Biochem (2014) 387:91–100

123

Hypoxic preconditioning of hUC-MSCs

P3 hUC-MSCs were incubated in DMEM/F12 medium

containing 100 lmol/l of cobalt chloride and 0.1 % FBS in

a humidified 5 % CO2 incubator at 37 �C for 48 h.

ELISA assay

P3 hUC-MSCs with or without hypoxic preconditioning

were trypsinized, counted, and plated in a 24-well plate at a

density of 2 9 104 per well, with six replicate wells for each

group. After adherence of MSCs to the wall surface, mito-

mycin-C of 25 lg/ml (MMC, Kyowa Hakko Kogyo, Japan)

was added into each well to mitotically inactivate MSCs.

Then hUCB-CD34? cells (2 9 104/well) and PBMCs

(2 9 105/well) suspended in RPMI-1640 were added and

cultured for 96 h at 37 �C in a 5 % CO2 incubator. For the

negative control, hUC-MSCs were omitted. The groups were

divided as follows: A1 = HP-MSCs ? hUCB-CD34?

cells ? PBMC, A2 = hUC-MSCs ? hUCB-CD34? cells ?

PBMC, Negative Control = hUCB-CD34? cells ? PBMC.

Culture supernatants of each group were collected, and the

IL-10 and IFN-c levels were measured with a ELISA

detection kit (Ever, USA). Each well was repeated twice

following the manufacturer’s instructions.

Experimental animals

A rabbit model of MI was established using a modified

Fujita method [15]. Adult female Japanese white rabbits,

weighing 2.57 ± 0.45 kg, were anesthetized by intramus-

cular injection of ketamine (25 mg/kg) and intraperitoneal

injection of 1 % pentobarbital sodium (1 ml/kg). A median

incision was made, and the left ventricular branch (LVB)

was ligated at the midpoint between the starting point of

the major branch and the cardiac apex with a 6–0 Prolene

suture. Myocardial ischemia was confirmed by both ST-

segment elevation on the ECG and regional cyanosis of the

myocardial surface. No drainage was performed. The ani-

mals were kept warm with a heating pad and allowed to

recover. The survived rabbits were administered with

80 mg/kg penicillin im QD for 3 days.

Cell transplantation

A second thoracotomy was performed 4 weeks after MI

following the same procedures as described above. The

animals were randomized into three groups and received

intramyocardial injections of 0.4 ml of PBS alone (n = 8,

PBS group), 5 9 106 hUC-MSCs in PBS (n = 8, hUC-

MSCs group), or 5 9 106 hUC-MSCs ? 5 9 105/kg

CD34? cells in PBS (n = 8, Cotrans group), at four points in

the infarct border zone.

Evaluation of heart function

Echocardiography was performed at baseline, 4 weeks

after MI induction, and 4 weeks after cell transplantation,

respectively.

Histopathological examination

The rabbits were euthanized by 10 % KCl f4 weeks after

cell transplantation. The hearts were excised, fixed in 10 %

formalin for [24 h, and cut transversely at the ligation.

Then the myocardial tissues below the ligation site were

embedded in paraffin and sectioned into 4- to 5-lm-thick

slices, which were to be used for hematoxylin and eosin

(H&E), Masson’s trichrome, and immunohistochemistry.

Immunohistochemical stain

For the immunohistochemical detection of CD31, the tissue

sections were incubated with the primary mouse mono-

clonal antibody to CD31 (1:15, Abcam, UK), followed by a

second incubation with HRP-conjugated goat anti-mouse

IgG antibody (Two-Step IHC Detection Reagent, ZSGB-

BIO, China). For the detection of cTnI, the sections were

incubated with the sheep polyclonal anti-cTnI antibody

(1:100, Abcam, UK) and then HRP-conjugated rabbit anti-

sheep IgG secondary antibody (1:500, CUSABIO, China).

At last, the tissue sections were stained with DAB.

Determination of vessel density

CD31-positive vessels were counted in five randomly

selected high-power fields under a light microscope at

2009 (Olympus, Japan), and the vessel density was defined

as the mean number blood of vessels.

Statistical analysis

All data were expressed as mean ± SE. Statistical analysis

was performed by one-way ANOVA, followed by LSD

post hoc test using SPSS version 19.0 (SPSS Inc., USA).

P \ 0.05 was considered statistically significant.

Results

Isolation and culture of hUC-MSCs

hUC-MSCs adhered to the plastic surface of the flask at the

first change of medium. 5 h after passage, some hUC-

MSCs adhered to the bottom of the flask in a spindle or

triangle shape (Fig. 1a). More adherent cells, primarily

Mol Cell Biochem (2014) 387:91–100 93

123

with a spindle morphology, were observed 24 h after pas-

sage (Fig. 1b), and grown to 80–100 % confluence in a

whirlpool-like or parallel array 5–6 days after inoculation

(Fig. 1c). Small and round CD34? cells were successfully

isolated from hUCB (Fig. 1d).

Immunophenotype of hUC-MSCs and purity of CD34?

cells

hUC-MSCs expressed high levels of CD90 and CD105, but

low levels of CD34 and CD45 (Fig. 2a). It is similar to BM-

MSCs which indicates that hUC-MSCs may have biological

characteristics similar to those of BM-MSCs. The purity of

CD34? cells was detected every time the cells were posi-

tively selected. After repeating ten times, the average purity

of CD34? cells was 93.89 ± 3.88 % (Fig. 2b).

ELISA array

Table 1 showed that IFN-c was 27.00 ± 1.11, 14.20 ±

0.81, and 7.22 ± 0.14 pg/ml for the Control, A1 and A2

groups, respectively. The results indicated that peripheral

blood lymphocytes could be activated to secrete IFN-c by

UCB-CD34? cells, and that both hUC-MSCs and HP-MSCs

significantly reduced IFN-c secretion (P = 0.04, n = 6). It

also showed that IL-10 was 31.68 ± 3.08, 61.42 ± 1.08,

and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups,

respectively, which indicated that both cells significantly

increased IL-10 secretion by lymphocytes (P = 0.03,

n = 6). A close comparison between A1 and A2 revealed

that HP-MSCs had a weaker immunomodulatory effect on

IFN-c and IL-10 secretion than hUC-MSCs (P = 0.03,

n = 6).

Heart function

As shown in Fig. 3, no significant difference was found in

baseline heart function among the groups. However, left

ventricular fractional shortening (LVFS) decreased signifi-

cantly in all animals 4 weeks after LVB ligation, and was

restored to baseline level four weeks after cell transplanta-

tion in both Cotrans and hUC-MSCs groups, but decreased

continuously in the PBS group (31.63 ± 2.20 vs. 40.13 ±

2.48 % for the Cotrans group, P = 0.000; 31.25 ± 2.12 vs.

36.25 ± 1.75 % for the hUC-MSCs group, P = 0.00;

32.75 ± 1.17 vs. 32.00 ± 0.76 % for the PBS group, P =

0.02, respectively). It was evident that cotransplantation of

hUC-MSCs and CD34? cells resulted in a significantly

higher LVFS than hUC-MSCs (P = 0.003) or PBS

(P = 0.00) and LVFS in hUC-MSCs are higher than that in

PBS (P = 0.000). The left ventricular end-systolic diameter

(LVESD) decreased in both hUC-MSCs and Cotrans group

Fig. 1 The morphology of hUC-MSCs and hUCB-CD34? cells.

a 5 h after passage, some hUC-MSCs adhered to the surface of

culture flask and appeared to be spindle-shaped or triangular. b 1 day

after passage, most of the hUC-MSCs adhered to the surface of

culture flask with typical fibroblast-like or spindle shapes. c 5 days

after passage, hUC-MSCs reached 80–100 % confluence in a vortex

or parallel array. d CD34? cells were isolated from hUCB success-

fully with a small and round morphology. Magnified 9100

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123

(9.46 ± 0.73 vs. 8.57 ± 0.52 for the hUC-MSCs group,

P = 0.036; 10.27 ± 0.49 vs. 8.31 ± 1.79 for the Cotrans

group, P = 0.03), as shown in Fig. 4, and this may explain

the increase in LVFS. There was a difference in the heart rate

between the Cotrans and PBS group, but with no practical

significance in this case.

Pathological changes of infarcted myocardium

An improved pathological response was observed in animals

treated with hUC-MSCs or cotransfection, as more cardio-

myocytes (red) survived and less collagen (blue) was

deposited in comparison with the PBS group. The Cotrans

group had the lowest collagen deposition in the peri-infarc-

ted area, as evidenced by HE and Masson’s trichrome

staining in Fig. 5.

Immunohistochemistry

Some cTnI-positive cells and CD31-positive tubular struc-

tures were present in the peri-infarcted area 4 weeks after

transplantation (Fig. 6), whereas no cTnI-positive cells in the

areas remote to the infarcted area. These indicated the dif-

ferentiation of stem cells into cardiomyocytes and neovascu-

larization. The cTnI-positive cells only exist in the Cotrans

group, which indicated that the new method of cotransplan-

tation can induce the cardiomyogenic differentiation.

Vessel density

The presence of CD31-positive tubular structures in the

peri-infarcted area could be interpreted as an indicator of

neovascularization in animals treated with stem cells

(Fig. 6). The microvessel density was 12.19 ± 3.05/HP for

the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans

group, respectively (P = 0.000). However, no CD31-

positive vessels were detected in the PBS group.

Fig. 2 Flow cytometric analyses. hUC-MSCs (2 9 105) in the third

passage (P3) were trypsinized, suspended in 200 ll PBS, and then

incubated for 30 min at room temperature with PE- or FITC-

conjugated mouse anti-human monoclonal antibodies (CD34, CD45,

CD90, and CD105). Mouse isotype antibodies served as controls. It

a is shown that hUC-MSCs expressed high level of CD90 and CD105,

but low level of CD34 and CD45. The purity of CD34? cells was

detected every time the cells were positively selected. Then average

the numbers after repeating ten times. b shows the representative

result

Table 1 Immunomodulatory effects of hUC-MSCs and HP-MSCs

Group n IFN-c (pg/ml) IL-10 (pg/ml)

A1 6 14.20 ± 0.81*,# 61.42 ± 1.08*,#

A2 6 7.22 ± 0.14* 85.85 ± 1.80*

Control 6 27.00 ± 1.11 31.68 ± 3.08

A1 = HP-MSCs ? hUCB-CD34? cells ? PBMC; A2 = hUC-

MSCs ? hUCB-CD34? cells ? PBMC; Control = hUCB-CD34?

cells ? PBMC

* P \ 0.05 compared to control; # P \ 0.05 compared to A2

Mol Cell Biochem (2014) 387:91–100 95

123

Discussion

In this study we showed that transplantation of hUC-MSCs

or hUCB-CD34? cells improved heart function in post-MI

rabbits, and that PBS-treated animals had a persistently

depressed left ventricular function. However, a combined

regimen of hUC-MSCs and hUCB-CD34? cells would be

more desirable than either cells alone. Clearly, our results

have important implications for stem cell-based therapy for

MI. In this study, MSCs were successfully isolated from

UC by enzyme digestion, and CD34? cells with a high

level of purity were positively selected from UCB by

immunomagnetic bead separation. The ELISA results

indicated that peripheral blood lymphocytes could be

activated by UCB-CD34? cells to secrete IFN-c, which

could modulate cell-mediated immunity and immune

rejection. However, a decreased IFN-c secretion and

increased IL-10 secretion were observed in rabbits cocul-

tured with hUC-MSCs. IL-10 has been reported to down-

regulate CD80 expression, disable T cells, and induce

immune tolerance [16]. The present study showed that

cotransplantation of hUC-MSCs and hUCB-CD34? cells

resulted in an improved immunological tolerance of

cardiomyocytes.

hUC-MSCs do not express HLA-class-II molecules and

express only a low level of HLA-class-I molecules [12],

indicating that hUC-MSCs have a low immunogenicity and

are immunoprivileged. In addition, hUC-MSCs do not

express costimulatory molecules such as CD40, CD80, and

CD86 and thus are unable to stimulate the proliferation of

human peripheral blood lymphocytes [13]. hUC-MSCs

secrete no IFN-c and little IL-10 (\7.8 pg/ml) [10, 17]. All

Fig. 3 Time course changes of LVFS at baseline, 4 weeks after LVB

ligation (preinjection), and 4 weeks after cell transplantation (post-

injection). Eight rabbits in every group were lightly anesthetized with

ketamine and pentobarbital before the evaluation by echocardiogra-

phy. *P \ 0.05; **P \ 0.01. #P \ 0.01 compared to PBS group;##P \ 0.01 compared to hUC-MSCs group

Fig. 4 Comparison of other parameters between preinjection and

postinjection. The LVESD decreased in both hUC-MSCs and Cotrans

group and this may explain the increase in LVFS. There was a difference

in the heart rate between the Cotrans and PBS group, but with no practical

significance in this case. a HR = heart rate, b LVEDD = left ventricular

end-diastolic dimension, c LVESD = left ventricular end-systolic

dimension, d LVPWDT = left ventricular posterior wall end-diastolic

thickness, and e LVPWST = left ventricular posterior wall end-systolic

thickness. *P \ 0.05

96 Mol Cell Biochem (2014) 387:91–100

123

Fig. 5 Representative pictures of H&E and Masson’s trichrome

staining. Animals in every group (n = 8) were euthanized by 10 %

KCl and the myocardial tissues below the ligation site were sectioned

into 4- to 5-lm-thick slices, Two slices of each heart were used for

staining. Magnified 9200. They came from the similar position of the

infarction border zone

Fig. 6 Representative pictures of IHC. a showed cTnI-positive cells

in the peri-infarcted area of Cotrans group, but no cTnI-positive cells

in the PBS and hUC-MSCs group. b There are no CD31-positive

vessels in the PBS group. The hUC-MSCs group c showed a less

degree of angiogenesis than Cotrans group d at 4 weeks after stem

cells transplantation. The capillary positive for CD31 staining was

counted in four high-power fields (9200). Then the average of the

four numbers of positive capillary was taken as the average capillary

density (ACD). The ACD in the Cotrans group was much higher than

hUC-MSCs group (**P \ 0.01), which indicated more significant

microvessel formation after Cotransplantation (g). e, f showed the

high-magnification (9400) view of the red boxes in the (c) and (d),

respectively

Mol Cell Biochem (2014) 387:91–100 97

123

these results indicate that hUC-MSCs have immunosup-

pressive properties. Thus, from an immunological per-

spective, it makes possible cotransplantation of hUC-MSCs

and hUCB-CD34? for the treatment of MI.

The ELISA results showed that the immunosuppressive

effect of the hypoxia-preconditioned hUC-MSCs was

attenuated, the underlying mechanism remains to be deter-

mined. In this regard, despite an enhanced secretion of pro-

angiogenic factors in response to hypoxic preconditioning

[11], cotransplantation of HP-MSCs and hUCB-CD34? cells

might not be a good choice for the treatment of MI.

Several animal studies have shown that BM-MSCs

could restore heart function after MI, decrease collagen

deposition, and ameliorate LV remodeling [2, 11, 18].

Nevertheless, there is a paucity of studies on the treatment

of MI with hUC-MSCs. Latifpour et al. [4] showed that

undifferentiated hUC-MSCs improved heart function after

MI and differentiated into cardiomyocytes in vitro. Our

results also showed that hUC-MSCs improved heart func-

tion after MI, with an increase of LVFS from 31.25 ± 2.12

to 36.25 ± 1.75 % and less collagen deposition. Cotrans-

plantation of hUC-MSCs and hUCB-CD34? cells resulted

in a higher LVFS and improved heart function as compared

with hUC-MSCs administered alone.

There has been an ongoing debate about the mechanisms

responsible for stem cell therapy for MI. It has been proved

that stem cells differentiate into cardiomyocytes in vitro

and in vivo [4, 19], but with an extremely low efficiency

[20]. In this study, immunohistochemical staining revealed

the presence of a great number of cTnI-positive cells in the

infarcted area of stem cell-treated animals. It is most likely

that hUC-MSCs interact with hUCB-CD34? cells that

enhances the transdifferentiation of stem cells to cardio-

myocytes. The exact mechanism that accounts for this is

still unknown, but it might be related to cytokines secreted

by MSCs that prevent early death of the stem cells and

promote their survival and proliferation. Williams et al.

[21] also showed that combining human cardiac stem cells

with hMSCs produced a greater infarct size reduction and

improved heart function as compared with either cells

administered alone, and it showed sevenfold enhanced

engraftment of stem cells in the combination therapy group

versus either cell type alone.

It is also argued that stem cells differentiate into endo-

thelial cells, resulting in an increase of vessel density in the

infarcted area, tissue reperfusion, and eventually improved

heart function [22, 23]. We found that there were more

CD31-positive microvessels in the Cotrans group than in

the hUC-MSCs group, suggesting that cotransplantation of

hUC-CD34? cells and hUC-MSCs has the potential to

increase neovascularization. Again, this is more likely due

to soluble cytokines secreted by stem cells, but not due to

differentiation of stem cells. Nevertheless, a more rigorous

test of this hypothesis is needed before a solid conclusion

can be drawn. MSCs expressed higher vascular endothelial

growth factor (VEGF) mRNA than hemopoietic progenitor

cells in BM [24], and the expression of VEGF and basic

fibroblast growth factor in the heart tissues of a swine

model of chronic MI was increased after infusion of BM-

MSCs [25]. Thus it is believed that paracrine function may

constitute the primary mechanism responsible for the stem

cell therapy for MI.

This study has important theoretical and applied impli-

cations for stem cell therapy in post-MI patients. Both

hUCB-CD34? cells and hUC-MSCs are easily accessible

without any invasive procedures and ethical problems. In

addition, mesenchymal stem cells have an immunosup-

pressive ability so that immuno-suppressant is not neces-

sary. hUC-MSCs have multipotency of differentiation into

various tissue cells, including chondrocytes [26], adipo-

cytes [27], and osteoblasts [28], and are, therefore, an ideal

candidate for cellular therapy. They have a shorter popu-

lation doubling time than BM-MSCs [29]. Recent advances

of stem cell biology make possible a more favorable

therapeutic outcome with the use of complementary cells.

Conclusions

Both hUC-MSCs and HP-MSCs have an immunosuppres-

sive effect on lymphocytes, which, however, can be attenu-

ated by hypoxic preconditioning. Cotransplantation of hUC-

MSCs and hUCB-CD34? cells can improve heart function

and decrease collagen deposition in post-MI rabbits. This is

most likely due to the increase of cardiomyocytes and

enhanced angiogenesis in the infarcted myocardium.

Acknowledgments This study was supported by the Natural Sci-

ence funds of Tianjin Province (10JCYBJC14000). The authors are

very grateful for the sincere help and excellent technical support by

the Key Laboratory of Artificial Cell, Institute of Hepatobiliary Dis-

ease of Tianjin Third Central Hospital.

Conflict of interest The authors declare that no conflicts of interest

exist.

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