Accepted Manuscript

Title: Human umbilical cord mesenchymal stem cells

transplantation improves cognitive function in Alzheimer’s
disease mice by decreasing oxidative stress and promoting
hippocampal neurogenesis

Author: YuanBo Cui ShanShan Ma ChunYan Zhang Wei Cao

Min Liu DongPeng Li PengJu Lv Qu Xing RuiNa Qu Ning
Yao Bo Yang FangXia Guan

PII: S0166-4328(16)31281-5
DOI: http://dx.doi.org/doi:10.1016/j.bbr.2016.12.021
Reference: BBR 10614

To appear in: Behavioural Brain Research

Received date: 26-8-2016

Revised date: 12-12-2016
Accepted date: 16-12-2016

Please cite this article as: Cui YuanBo, Ma ShanShan, Zhang ChunYan, Cao
Wei, Liu Min, Li DongPeng, Lv PengJu, Xing Qu, Qu RuiNa, Yao Ning, Yang
Bo, Guan FangXia.Human umbilical cord mesenchymal stem cells transplantation
improves cognitive function in Alzheimer’s disease mice by decreasing oxidative
stress and promoting hippocampal neurogenesis.Behavioural Brain Research
http://dx.doi.org/10.1016/j.bbr.2016.12.021

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Human umbilical cord mesenchymal stem cells transplantation
improves cognitive function in Alzheimer’s disease mice by
decreasing oxidative stress and promoting hippocampal neurogenesis

YuanBo Cuia,1, ShanShan Mab,1, ChunYan Zhangc, Wei Caoa, Min Liua,
DongPeng Lid, PengJu Lv a, Qu Xingb, RuiNa Qub, Ning Yaob, Bo Yangd,*and
FangXia Guanb,*
a
Translational Medicine Center, Zhengzhou Central Hospital Affiliated To Zhengzhou University,

Zhengzhou 450007, People’s Republic of China; [email protected](Y.B.-C.);

[email protected](W.-C.); [email protected](M.-L.);[email protected](P.J.-L)
b
School of Life Sciences, Zhengzhou University, Zhengzhou 450001, People’s Republic of China;

[email protected](S.S.-M); [email protected](Q.-X.); [email protected](R.N.-Q.);

[email protected](N.-Y.)
c
Department of Laboratory，Zhengzhou Central Hospital Affiliated To Zhengzhou University,

Zhengzhou 450007, People’s Republic of China; [email protected] (C.Y.-Z)

d
Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou

450052, People’s Republic of China

*
Corresponding authors: Fangxia Guan, [email protected]; Bo Yang, [email protected]

1
These authors contributed equally to this work.
Highlights

 In the present study, we first examined whether the intravenous

transplantation of stem cells could have beneficial effects in

Tg2576 AD mice, and further investigated the mechanism of action

by which hUC-MSCs might improve cognitive function.

 Our findings demonstrate that hUC-MSCs can improve cognition

of AD mice by decreasing oxidative stress and promoting

hippocampal neurogenesis and accompanied with increased protein

levels of Sirt1,BDNF and SYN.

Abstract

Stem cell transplantation represents a promising therapy for central nervous system
injuries, but its application to Alzheimer’s disease (AD) is still limited and the
potential mechanism for cognition improvement remains to be elucidated. In the
present study, we used Tg2576 mice which express AD-like pathological forms of
amyloid precursor protein (APP) to investigate the effects of human umbilical cord
mesenchymal stem cells (hUC-MSCs) intravenous transplantation on AD mice.
Interestingly, hUC-MSCs transplantation significantly ameliorated cognitive function
of AD mice without altering Aβ levels in hippocampus. Remarkably, hUC-MSCs
transplantation reduced oxidative stress in hippocampus of AD mice by decreasing the
level of malondialdehyde (MDA), increasing the level of nitric oxide (NO), enhancing
the activity of superoxide dismutase (SOD) and neuronal nitric oxide synthase
(nNOS). The mechanisms underlying the improved cognitive function may be linked
to hippocampal neurogenesis and an up-regulation of neuronal synaptic plasticity
related proteins levels including silent information regulator 1 (Sirt1), brain-derived
neurotrophic factor (BDNF) and synaptophysin (SYN). Taken together, our findings
suggest that hUC-MSCs can improve cognition of AD mice by decreasing oxidative
stress and promoting hippocampal neurogenesis. These results suggest that
modulating hUC-MSCs to generate excess neuroprotective factors could provide a
viable therapy to treat AD.

Keywords: human umbilical cord mesenchymal stem cells; Alzheimer’s disease;

cognitive function; oxidative stress; hippocampal neurogenesis
1. Introduction

Alzheimer’s disease (AD) is the most common form of dementia occuring

predominantly after 65 years of age and is clinically characterized by the progressive
cognitive dysfunction, memory loss and behaviour deficit (Querfurth,2010;
Thies,2011). Pathologically, senile plaque deposition composed of β-amyloid (Aβ) ,
neurofibrillary tangles and neuronal loss in cortex and hippocampal areas associated
with cognitive function decline are three representative hallmarks of AD (Selkoe,2001;
Holtzman,2011). Currently, there has been no effective therapy for AD
(Cummings,2014; Holtzman,2011; Miller,2010). The number of AD patients is
expected to rise to over 100 million by 2050 according to the World Alzheimer report
(Alzheimer's Association, 2010). Therefore, it is critical to develop an effective
treatment to improve cognition and alleviate pathology of AD.
Stem cell-based therapy has emerged as a promising potential approach to treat
AD. In recent years, accumulating evidence indicates that transplantation of adult
stem cells such as bone marrow stem cells (BMSCs), adipose mesenchymal stem cells
(ADMSCs) or neural stem cells (NSCs) into the lateral ventricles could ameliorate
cognition deficits in AD animal models (Blurton,2009; Lee,2015; Yan,2014;
Babaei,2012; Ma,2013). However, harvesting these types of stem cells is fraught
with many problems, such as low yield, ethical issues, and invasive procedure.
Additionally, using stereotactic techniques to transplant stem cells into brain causes
damage, thus, not making this technique suitable for patients. Therefore, these
shortcomings limit the application for clinical usage. Human umbilical cord
mesenchymal stem cells (hUC-MSCs) derived from Wharton’s jelly of human
umbilical cord have attracted more attention because it is a noninvasive procedure,
sufficient cell number and no ethical problem. In addition, hUC-MSCs possess greater
paracrine effects and increased cell proliferation and differentiation into neurons
compared to BMSCs and ADMSCs (Yang, 2013; Shih,2015; Zhou,2011;
Toupadakis,2010; Zohreh,2015). Although the therapeutic effects of stereotactic
delivery of stem cells on AD mice have been demonstrated, the effect of intravenous
transplantation of stem cells, especially hUC-MSCs, in a noninvasive manner on AD
models is controversial, and the underlying mechanism for cognition improvement
remains to be elucidated.
It has been reported that when stem cells are administered via the intravenous
route, they could cross the blood-brain barrier (BBB) and migrate to the hippocampus
and improve cognition associated with decreased Aβ deposition (Kim,2012). In
contrast, other results have demonstrated that intravenous transplantation stem cells
could ameliorate the decline in cognition without altering Aβ burden by promoting
synaptic plasticity (Zhang,2014). However, some researchers have found that
intravenous administration of stem cells did not cross the BBB and had no beneficial
effect for AD (Ehrhart,2016). Recent evidence supports the notion that intravenous
transplantation of stem cells can migrate to many organs from one week until three
months (Kim,2013; Park,2016). However, the therapeutic effect of intravenous stem
cells administration on AD mice and the mechanistic basis of the observed
improvement has not be determined.
In the present study, we first examined whether the intravenous transplantation of
stem cells could have beneficial effects in Tg2576 AD mouse model, and to further
investigate the mechanistic basis by which hUC-MSCs improve cognitive function.

2. Experimental procedures

2.1. Animals

Tg2576 mice, on a C57BL/6 background, which harbors mutant human gene APPswe
(Swedish mutations K670N/M671L) under the control of mouse prion protein
promoter, were obtained from the Institute of Laboratory Animal Science, Chinese
Academy of Medical Science (Beijing, China). The positive APP transgenic mice
were identified by PCR test before this study. Because of gender-specific differences
in the progression of Aβ deposition, only male mice were used in this study. All
animal procedures were performed following the National Institutes of Health
Guidelines for the Humane Treatment of animals and in accordance with the Ethical
Committee for Animal Experiments of Zhengzhou University. Mice were housed
under a 12h light/dark cycle and automatically maintained at 22-25℃ and humidity,
allowed standard rodent chow and water ad libitum. This research was a single blind
study.

2.2. Isolation and transplantation of hUC-MSCs

Human umbilical cords were obtained from full-term infants delivered after caesarian
section with the written informed consent of the parents. The procedure was approved
by the ethical committee of the Zhengzhou Central Hospital Affiliated to Zhengzhou
University and based on the previous description by Zhou and Xie et al (Zhou,2011;
Xie,2016). After washing residual blood with Hank’s balanced salt solution and the
removal of vessels and umbilical cord membrane, the mesenchymal Wharton’s jelly
was cut into about 1-cm3 pieces and suspended in Dulbecco’s modified Eagle
media/F12 (DMEM/F12; GIBCO,USA) supplemented with 10% fetal bovine serum
(FBS;GIBCO,USA) ,100 U/ml penicillin, 100 U/ml streptomycin, and 20ng/ml basic
fibroblast growth factor (bFGF; Sigma, USA). The tissue suspension culture system
was left undisturbed for 7 days in 5 % CO2 in a 37℃ incubator to allow cells to
migrate from the explants. The culture medium was replaced every 3 days.
hUC-MSCs were passaged when they reached 80% confluence by using 0.25 %
trypsin-EDTA solution. Cells were analyzed by flow cytometry to confirm
immunophenotype of hUC-MSCs as previously described (Zohreh,2015;
Shanshan,2014).
Twelve-month-old Tg2576 mice were randomly divided into three groups: Wild
type (WT) (n=15), vehicle-treated AD (n=15), and hUC-MSCs (n=15). For WT group,
we used age-matched non-transgenic C57BL/6 mice. For vehicle-treated AD group,
we injected 200μL vehicle, phosphate buffered saline (PBS). For hUC-MSCs group,
we injected in the right tail vein with 200μL hUC-MSCs cell suspension
(approximately 2×106 cells). No mouse died and there was no aberrant cell growth or
tumor formation during the period of study.
2.3. BrdU injection

Mice were injected intraperitoneally with 5-bromodeoxyuridine (BrdU) (50 mg/kg;

Sigma) once a day for 7 days after hUC-MSCs transplantation to identify the degree
of newborn cell survival in the hippocampus by using immunohistochemical
identification.

2.4. Morris Water Maze test

Four weeks after cell transplantation, the cognitive performance of mice was assessed
by Morris Water Maze (MWM) test and based on the previously described method
(Ruzicka,2016;Li,2013). The MWM consisted of a circular pool(height 40cm,
diameter 120cm, four quadrants) filled with water(containing milk,24-26℃) and an
escape platform (height 28 cm, diameter 10cm) was positioned submerged 0.5-1cm
below the surface of the water. The first stage is the training period in order to allow
mice to adapt to the surrounding environment, training twice a day for six consecutive
days. Probe trial and navigation test were conducted on the seventh day and each
mouse was allowed to swim only up to 60 second until it sought the escape platform.
Motion parameters of each mouse were recorded in computer program. A consistent
quiet environment was maintained throughout the period of the experiment.

2.5. Tissue preparation

Two mice in both vehicle-treated AD group and hUC-MSCs group were sacrificed at
7d and 28d after hUC-MSCs transplantation for PCR test and anti-MAB1281
immunostaining. The other mice were sacrificed after MWM test or at the appropriate
time. The mice were anesthetized with pentobarbital (Sigma) (50 mg/kg) and
immediately cardiac-perfused with 0.9 % saline solution. For morphological analysis,
one cerebral hemisphere or hippocampus was fixed in 4 % paraformaldehyde solution
overnight and incubated in 30% sucrose solution for 3 days at 4℃ until equilibration
(5 mice per group) and then sequential 25- or 10-μm coronal sections were cut on a
cryostat (Cryotome, Thermo Fisher) and stored at -20℃. For biochemical and
molecular biological analysis, the other half cerebral hemisphere or hippocampus was
immediately frozen in liquid nitrogen. Frozen tissues were homogenized in T-PER
regent (Thermo Fisher) with protease inhibitors (Thermo Fisher) by using a
ultrasound homogenizer (Thermo Fisher). Supernatant was collected after centrifuged
for 1h at 14000g, 4℃. The sediments were resuspended in 70% formic acid solution
and then centrifuged for 1h at 14000g, 4℃ to collect the supernatant to detect the
insoluble Aβ40,42.

2.6. Polymerase chain reaction analysis

In order to confirm that hUC-MSCs could migrate to brain and remained for an
extended period after a single intravenous injection, the presence of human specific
DNA in the hippocampus of Tg2576 mice was detected at 7d, 28d after administration
by using PCR technique and agarose gel electrophoresis(AGE). Total purified DNA
was extracted from brain tissues using a QIAamp DNA Blood/tissue Kit (Qiagen,
USA) and quantified by a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA).
Polymerase chain reaction (PCR) technique (total volume 50μL) was used to detect
the presence of human specific DNA in mice brain tissues(Liu,2014). PCR products
were detected by 1% agarose gel (Invitrogen, USA) electrophoresis, the gel was
stained with GelRed (Sigma, USA) and visualized using UV translumination. Sense
primer: 5’-GGGATAATTTCAGCTGACTAAACAG-3’, antisense primer:
5’-AAACGTCCACTTGCAGTTCTAG-3’.

2.7. Aβ ELISA measurement

Hippocampus samples were stored in liquid nitrogen until homogenization. Soluble

and insoluble Aβ40 and Aβ42 levels were quantified by using Aβ40,42 ELISA kits
(Invitrogen, NY). The assay was performed according to the manufactuer’s
instruction. Soluble Aβ40,42 in hippocampus homogenates was detected at 1:10
dilutions and insoluble Aβ40,42 in hippocampus homogenates was detected by
extracting sediments in 5 mol/L guanidine-HCl at 1:20 dilutions in lysis buffer.

2.8. γ-secretase activity measurement

Aβ-generating enzyme γ-secretase activity in hippocampus homogenates was

measured by using a γ-secretase activity kit (R&D systems, MN) and the procedure
was according to the manufactuer’s instruction.

2.9. Determination of oxidative markers

Homogenates supernatant of hippocampus samples were used to detect the relative

level of oxidative markers including the activity of superoxide dismutase (SOD) and
neuronal nitric oxide synthase (nNOS), the levels of malonaldehyde (MDA) and total
nitric oxide (TNO) by using commercial kits(Jiancheng Bioengineering Inst., China).
Protein content in supernatant was quantified by a Nanodrop 2000 spectrophotometer
(Thermo Fisher, USA). The procedure was according to the manufactuer’s instruction.

2.10. Immunohistochemical staining

Immunohistochemical staining for BrdU was used to detect the degree of surviving
newborn and immature cells in hippocampus. And, MAB1281 positive staining was
used as a marker for exogenous hUC-MSC. For each mouse, six coronal sections
(each 10-μm thick) throughout the hippocampus were analyzed. All sections were
incubated in 1 mol/L HCl for 30min at room temperature, then washed with borate
buffer for 15 min and washed with phosphate buffered saline three times for 10 min.
Followed by blocked with 5 % goat serum for 30 min and then incubated with rabbit
anti-BrdU primary antibody (recognizing newborn cells, Santa Cruz, USA; diluted in
1:1000) or anti-human nuclei primary antibody MAB1281 (recognizing human cells,
Santa Cruz, USA; diluted in 1:2000) overnight at 4℃. Washing with PBS and
incubating with secondary antibody (HRP-conjugated anti-goat-IgG, diluted in 1:500)
in blocking buffer for 1 h at room temperature. Finally, sections were incubated with
DAB and mounted on glass slides. All positive cells in the subgranular zone of the
contralateral dental gyrus (DG) of hippocampus were counted and averaged.

2.11. Nissl staining

After MWM test, Nissl staining was performed to evaluate the effect of
transplantation hUC-MSCs on neuronal regeneration. For each mouse, six coronal
sections (each 10-μm thick) were stained with 1% toluidine blue for 30 min at 56℃,
washed, color separated, dehydrated, hyalinized, and mounted by microscope.
Quantification was performed as previously described method (Chen, 2014). Cells in
sections that contained the hippocampus with a clear body and nucleus were counted
to analyze the levels of neurons (Zeiss, Germany).

2.12. Fluorescent immunohistochemical staining

Fluorescent immunohistochemical staining for BrdU and NeuN was used to detect the
degree of newborn neuronal cells in hippocampus. For each mouse, six coronal
sections (each 25-μm thick) throughout the hippocampus were analyzed. Sections
were denatured in 2 N HCl for 30 min at room temperature and rinsed in 100 mM
boric acid (pH 8.0) for 10 min, and then washed in 100 mM PBS. Sections incubated
in 100 mM PBS containing 10 % normal donkey serum and 0.25% Triton X-100 for 1
h at room temperature, and subsequently incubated with primary antibodies overnight
at 4℃, including BrdU (Santa Cruz, USA; diluted in 1:250), NeuN (Millipore, USA;
diluted in 1:1000). The next day, sections were washed in PBS and incubated with the
secondary antibodies including Cy3-conjugated donkey anti-rabbit(Jackson
ImmunoResearch, USA; diluted in 1:500), Alexa Fluor 647-conjugated donkey
anti-rat (Jackson Immuno Research, USA; diluted in 1:500) for 1 h at room
temperature. After washed with PBS and mounted on glass slides, sections were
evaluated for fluorescence on a confocal microscope (Olympus, USA).
2.13. Western blot analysis

Total proteins were extracted from hippocampus tissues after grinding with liquid
nitrogen in RIPA buffer with PMSF (Solarbio, China). Denatured protein (100 μg) of
each sample were separated by SDS-PAGE and transferred to a PVDF membrane
(Solarbio, China). The membrane was blocked with 5% skim milk powder in Tris
buffered saline tween (TBST) for 1 h. The protein blot was then incubated with
diluted primary antibodies and HRP-conjugated secondary antibody separately.
Immunoreactive proteins were detected by using the Chemidoc EQ system (BioRad,
USA). Primary antibodies used were: anti-Neprilysin (1:1000, Santa cruz), anti-IDE
(insulin degrading enzyme,1:1500, Santa cruz), anti-BACE1 (1:1000, Santa cruz),
anti-BDNF(1:2000,Thermo Fisher), anti-Sirt1(1:1000,Thermo Fisher),
anti-SYN(1:1000,Thermo Fisher) and anti-β-Actin (1:5000,Thermo Fisher).

2.14. Statistical analysis

The data were analyzed by using the Graphpad Prism 5 program (Graph Pad Software,
Inc., USA). Data are presented as Mean±Standard error of measurement (SEM).
Statistical significance was performed on the data by using one-way analysis of
variance (ANOVA) or unpaired student’s t-test. A value of P＜0.05 was considered to
be statistically significant.

3. Results

3.1. Morphologic and immunophenotypic characteristics of hUC-MSCs

hUC-MSCs were observed growing out from the mesenchymal Wharton’s jelly
fragments about one week after isolation, and displayed fibroblast-like
morphology(Fig. 1A-C). After immunophenotypic characteristics analysis, the cells in
this study were positive for CD73, CD90, and CD105, high levels of CD44 ,CD29
and low level of HLA-ABC, but negative for CD34, CD45, and HLA-DR, which was
consistent with hUC-MSC characteristics(Fig. 1D).

Figure1 The morphology and immunophenotypic characteristics of hUC-MSCs. (A)

hUC-MSCs grew out from the tissue pieces after cultured for one week (×200). (B-C) Passage-2

hUC-MSCs displayed to be mostly spindle-shaped or fibroblast-like morphology after 2days(B)

and 3dys(C) in culture(×200). (D) The morphology and immunophenotypic characteristics of

passage-3 hUC-MSCs.

3.2. Human specific DNA and human cells were observed in the hippocampus
after hUC-MSCs intravenous transplantation

The human DNA and human cells were observed in the brain within 7 days and
remained at 28d after hUC-MSCs transplantation (Supplementary Figure 1),
indicating that hUC-MSCs can migrate to the hippocampus within 7 days and remain
for an extended period of time.

3.3. hUC-MSCs transplantation improved cognitive function in Tg2576 mice

MWM test was performed to examine the cognitive function of the mice four weeks
after transplantation of hUC-MSCs. The complete protocol is summarized (Fig. 2A).
As expected, the vehicle infused Tg2576 mice exhibited dramatic impairments in the
MWM test compared with WT mice. However, the hUC-MSCs infused mice
performed significantly better than vehicle infused counterparts (p＜0.01; Fig. 2B), as
indicated by significantly shorter latency, higher proportion of time spent in the target
quadrant(Fig. 2C,E), and more crossing numbers (Fig. 2D).

Figure2 hUC-MSCs intravenous transplantation ameliorated the cognition dysfunction in

Tg2576 mice. (A) We transplanted the hUC-MSCs into 12-month-old Tg2576 mice and then

measured cognitive function four weeks after hUC-MSCs infusion. (B) Representative swimming

trajectories at day 7 of MWM test. (C) The data revealed that hUC-MSCs infused Tg2576 mice

exhibit significantly shorter escape latency on day 7 compared with vehicle infused AD mice

(ANOVA,P＜0.05). (D, E) hUC-MSCs transplantation restored spatial memory in the Tg2576

mice. Mice spatial memory was evaluated in a single probe test on day 7 after hUC-MSCs

infusion by the number of platform crossing (D) and the proportion of time spent in the target

quadrant (E). n=8 in each group. Data are presented as mean±SEM. ANOVA, *P＜0.05 and **P
＜0.01 compared with vehicle infused Tg2576 mice, #P＜0.05 compared with WT mice.

3.4. hUC-MSCs transplantation did not significantly affect levels of Aβ and

Aβ-degrading/ generating factors in hippocampus of Tg2576 mice

In order to examine the effect of hUC-MSCs intravenous transplantation on Aβ levels,

we tested the levels of both soluble and insoluble Aβ40,42 by using ELISA. The levels
of soluble Aβ40,42 and insoluble Aβ40,42 decreased in hippocampus of hUC-MSCs
infused mice, but there had no significant difference when compared to the vehicle
infused mice(P＞0.05,Fig. 3A-B). In order to examine the effect of the time point on
Aβ levels, we also detected the levels of soluble Aβ40,42 and insoluble Aβ40,42 in
hippocampus of mice two weeks and eight weeks after transplantation of hUC-MSCs.
Interestingly, our results showed that the levels of soluble Aβ40,42 and insoluble Aβ40,42
had no significant difference between the vehicle and hUC-MSCs infused mice
(Supplementary Figure 2). Next, we examined the activity of Aβ-generating enzyme
γ-secretase, the level of BACE1 and Aβ-degrading enzymes Neprilysin (NEP), Insulin
degrading enzyme (IDE), Matrix metalloproteinase-9 (MMP9). However, γ-secretase
activity and BACE1 level decreased in hippocampus of hUC-MSCs infused mice, but
showed no significant difference compared with vehicle infused mice(P＞0.05,Fig.
3C-D). The expression of NEP and IDE were elevated in hippocampus of hUC-MSCs
infused mice, but had no significant change when compared with the vehicle infused
mice(P＞0.05,Fig. 3D). These findings indicate that hUC-MSCs-induced cognitive
improvement might be involved in other regulatory pathways without modulating the
expression of Aβ degrading/ generating factors.
Figure3 hUC-MSCs intravenous transplantation has no significant effect on levels of Aβ and

Aβ-degrading/generating factors. (A,B) Soluble and insoluble Aβ40,42 levels were measured by

ELISA in the hippocampus. (C) The activity of γ-secretase was carried out by using a kit

according to the users manufacture, showed no significant difference between vehicle infused

group and hUC-MSCs group(P＞0.05). (D) In hippocampus, the expression of BACE1, NEP,

MMP9 and IDE were unaltered by infused hUC-MSCs(P＞0.05). n=5 in each group. Data

presented as mean±SEM. ANOVA, #P＜0.05 compared with WT mice.

3.5. hUC-MSCs transplantation attenuated oxidative stress in hippocampus of

Tg2576 mice

In order to confirm the beneficial antioxidative effect of hUC-MSCs on Tg2576 mice,

we examined the levels of oxidative makers in hippocampus homogenates. Our results
showed that the levels of MDA was significantly reduced (P＜0.05,Fig. 4A), the
levels of TNO and the activity of nNOS and SOD were significantly elevated (P＜
0.05,Fig. 4B-D) in the hippocampus of hUC-MSCs infused mice compared with
vehicle infused mice, suggesting that hUC-MSCs infusion resulted decreased
oxidative stress in the hippocampus of Tg2576 mice.

Figure4 hUC-MSCs intravenous transplantation decreased oxidative stress in hippocampus

of Tg2576 mice. (A) The level of MDA was reduced in hUC-MSCs infused mice. (B-D) The

activity of SOD and nNOS and the level of TNO in the hippocampus increased significantly in

hUC-MSCs infused mice when compared to vehicle infused mice(P＜0.05). n=5 in each group.

Data are presented as mean±SEM. ANOVA, *P＜0.05 compared with the vehicle infused

Tg2576 mice, #P＜0.05 compared with WT mice.

3.6. hUC-MSCs transplantation promoted cell proliferation and newborn cell

survival in hippocampus of Tg2576 mice

In order to determine the effects of intravenous transplantation hUC-MSCs on

hippocampal cell proliferation and newborn cell survival, we quantified BrdU positive
cells by anti-BrdU immunostaining on day 36. We found that there was a significant
reduction of BrdU positive cells in the hippocampus of vehicle infused Tg2576 mice
when compared to the WT mice(P＜0.05,Fig 5A-B). However, we also found that the
number of BrdU positive cells were elevated in the hippocampus of hUC-MSCs
infused Tg2576 mice when compared to the vehicle infused mice(P＜0.05,Fig 5B-C).
This suggests that the inhibition of cell proliferation in the hippocampus of Tg2576
mice was attenuated by hUC-MSCs transplantation.

Figure5 Immunochemistry of BrdU positive cells in hippocampus. Brain sections were

immunostained to measure BrdU positive cells by using anti-BrdU antibody. BrdU positive cells

represent cell proliferation and newborn cell survival. (A) BrdU positive cells in the DG of WT

mice. (B) BrdU positive cells in the DG of vehicle infused mice. (C) BrdU positive cells in the DG

of hUC-MSCs infused mice. (D) Computer aided image analysis of the BrdU positive cells in

hippocampus. Data are presented as mean±SEM. n=5 in each group. ANOVA, **P＜0.01

compared with the vehicle infused Tg2576 mice, #P＜0.05 compared with WT mice. Scale

bar=100μm. Bar graphs represent the mean number of BrdU-positive cells. Arrows indicate

BrdU-positive cells.

3.7. hUC-MSCs transplantation promoted neurons generating in hippocampus of

Tg2576 mice
In order to determine the effects of intravenous transplantation hUC-MSCs on
hippocampal neurogenesis, we used Nissl staining to observe survival neurons in
hippocampus on day 36. Our results showed neurons rich in Nissl bodies in the
cytoplasm at higher magnification (Fig. 6A). However, Nissl bodies in hippocampal
neurons of vehicle infused Tg2576 mice were significantly deceased or disappeared
and the neurons exhibited significant damage including loss of integrity (Fig. 6B). In
contrast, the number of neurons were significantly increased and the pyramidal cell
layer was densely arranged in hippocampus of hUC-MSCs infused Tg2576 mice (Fig.
6C), indicating that hUC-MSCs promotes neurons generating.

Figure6 Nissl staining of neurons in hippocampus. (A) Neurons with rich Nissl bodies in

cytoplasm in the DG of hippocampus in WT mice. (B) Nissl bodies were decreased or disappeared

in cytoplasm and the pyramidal cell layer was broken in the DG of vehicle infused mice. (C) The

number of neurons was dramatically increased in hUC-MSCs infused mice. (D) Quantitative

analysis of the number of neurons in hippocampus. Data presented as mean±SEM. n=5 in each

group. ANOVA, **P＜0.01 compared with the vehicle infused Tg2576 mice, #P＜0.05 compared

with WT mice. Scale bar=100μm. Bar graphs represent the mean number of neurons. Arrows
indicate neuronal cells.

3.8. hUC-MSCs transplantation promoting hippocampal neurogenesis of Tg2576

mice

In order to examine the effects of intravenous transplantation hUC-MSCs on

hippocampal neurogenesis, we used BrdU and NeuN double-labeling methods to
observe newborn neurons in hippocampus on day 36. The number of BrdU+/NeuN+
cells in the hippocampus of hUC-MSCs infused Tg2576 mice were significantly
increased compared with the vehicle infused mice(P＜0.01, Fig. 7A-C). Theses
results suggest that hUC-MSCs transplantation promoting hippocampal neurogenesis
of Tg2576 mice.

Figure7 Fluorescent immunohistochemical staining of newborn neurons in hippocampus.

(A-B) The hUC-MSCs infused Tg2576 mice showed increased BrdU+(red)/NeuN+(blue) cells in

the DG of the hippocampus. (C) Quantification of BrdU+/NeuN+ cells in hippocampus. Data

presented as mean±SEM. n=5 in each group. ANOVA, **P＜0.01 compared with the vehicle

infused Tg2576 mice. Scale bar=100μm. Bar graphs represent the mean number of neurons.

Arrows indicate newborn neuronal cells.

3.9. hUC-MSCs transplantation increased the expression of Sirt1, BDNF and
SYN in the hippocampus of Tg2576 mice

To further investigate the mechanism underlying the role of hUC-MSCs in promoting

neurogenesis, we examined the expression of neuroprotection related factors, Sirt1,
BDNF and SYN in the hippocampus by western blot. We found that the expression of
Sirt1, BDNF and SYN were dramatically decreased in the hippocampus of vehicle
infused mice when compared to WT mice (P＜0.05, Fig. 8A). Interestingly, these
three protein levels were significantly elevated in the hippocampus of AD mice after
infusion with hUC-MSCs (P＜0.05,Fig. 8B). Our findings suggest that increased
levels of Sirt1, BDNF and SYN could have beneficial effects on neurogenesis and
synaptic plasticity which affected cognitive improvement.

Figure8 Western blot were used to examine the expression of neuroprotective factors. (A)

Western blot analysis was performed to detect the expression of BDNF, Sirt1 and SYN in the

hippocampus of each group. (B) Protein levels were normalized with β-actin for quantity. Data

presented as mean±SEM. n=5 in each group. ANOVA, *P＜0.05 compared with the vehicle

infused Tg2576 mice, #P＜0.05 compared with WT mice.

4. Discussion

It has recently been suggested that stem cell treatment could have the potential
for the treatment of AD (Blurton,2009; Yue,2015; Hunsberger,2016). In the present
study, we used Tg2576 APP transgenic mouse, to detect whether intravenous
transplantation of hUC-MSCs could ameliorate cognition and to investigate the
underlying mechanism. We found that hUC-MSCs transplantation improved cognitive
function and this was associated with a reduction in markers of oxidative stress and
promoting neurogenesis in hippocampus of 12-month-old Tg2576 mice. However,
others suggested that intravenous or arterial transplantation of hUC-MSCs did not
have any beneficial effect on pathology of AD because the transplanted stem cells
could not migrate to brain (Ehrhart,2016; Khabbal,2015). Recent evidence indicates
that the intravenously transplanted stem cells cross the BBB, migrate to brain and
improve the cognition in the setting of AD (Kim,2012; Kim,2013; Park,2016;
Kim,2015). In our result, we observed the human specific DNA and human cells in
hippocampus of AD mice one week and four weeks after transplantation using PCR
technology and immunostaining, which enhanced the above points.
The MWM test results revealed that the vehicle infused Tg2576 mice exhibits
cognitive dysfunction and this impairment is significantly alleviated after intravenous
transplantation of hUC-MSCs, thus, strongly suggesting that the intravenous
transplantation of hUC-MSCs ameliorates AD. Next we examined the effect of
hUC-MSCs on Aβ pathology. In contrast to some studies which suggested that stem
cells could ameliorate the pathology of AD by decreasing Aβ levels in the form of
intravenous or cerebral transplantation (Xie,2016; Ruzicka,2016; Yang,2013).
However, consistent with some findings (Blurton,2009; Zhang,2014; Xuan,2009;
Rahasson,2015; Park,2016). Our results indicate that hUC-MSCs transplantation
could not significantly alter the Aβ changes. It is possible that the mice used in the
study were 12-month old mice with massive well-established Aβ deposition, which
could not be significantly eliminated by transplanted hUC-MSCs, and other
compensation mechanisms. These results suggest that the action mechanism of
hUC-MSCs improving cognitive function involves other pathways.
Previous studies reported that an increased number of degenerating neurons and
neuronal loss in the hippocampus is closely correlated with cognitive dysfunction
associated with AD, and excessive oxidation levels may exacerbate this impairment
(Sultana,2010; Tramutola,2016). Therefore, we determined the activities of SOD
and nNOS and the levels of MDA and TNO. Our results indicate that hUC-MSCs
decreases levels of MDA and increases levels of TNO and the activity of SOD and
nNOS in hippocampus of Tg2576 mice. These data indicate that hUC-MSCs
decreases oxidative stress, which consistent with previous reports (Yang,2013;
Chiang,2016). Interestingly, recent studies has demonstrated that NO plays a critical
role in synaptic plasticity of AD patients (Chakroborty, 2015; Kwon,2016).Thus, our
data added much more authority to these studies.
Results of Nissl staining and anti-BrdU immunostaining revealed that the
number of survival neurons and newborn cells were increased in hippocampus of
Tg2576 mice when compared to the vehicle infused mice, while many damaged
neurons and newborn cells were observed in hippocampus of vehicle infused Tg2576
mice. In the present study, we also observed the significantly increased number of
newborn neurons identified as BrdU(+)/NeuN(+) cells in hippocampus of Tg2576
mice after hUC-MSCs transplantation. These data demonstrate that the transplantation
of hUC-MSCs promotes neurogenesis in the hippocampus of Tg2576 mice.
To further investigate the underlying mechanism, we examined the protein level
of Sirt1, BDNF and SYN, which correlates closely with antioxidation, synaptic
connectivity and neurogenesis. We found that levels of Sirt1, BDNF and SYN
decreased significantly in Tg2576 mice when compared to WT mice. However, these
three proteins increased significantly in hippocampus of Tg2576 mice after
hUC-MSCs transplantation. Recent evidence supports the notion that Sirt1 plays an
important role in antioxidation and maintaining neuronal health and is essential for
normal cognitive function and synaptic plasticity (Michán,2010; Cho,2015;
Ming,2015). Currently, BDNF is known as a critical factor which correlated closely
with various neurological disorders such as Parkinson's disease and AD. In addition,
recent reports suggest that BDNF can enhance axonal outgrowth and is sufficient to
mediate cognitive recovery (Blurton,2009; Gottschalk,1999; Abigail,2015;
Janna,2016; Tanja,2015). Interestingly, it has been reported that the level of Sirt1
contributes to BDNF expression (Loredana,2012), suggesting that Sirt1 has a
synergetic effect with BDNF on neurogenesis. Growing evidence demonstrates that
the presynaptic protein SYN is essential for synaptic plasticity and synaptic
connectivity (Kwon,2011; Tampellini,2010). Thus, our results indicate that
hUC-MSC-induced increasing of Sirt1, BDNF and SYN mediates the improvement of
cognitive function in Tg2576 APP transgenic mice by promoting hippocampal
neurogenesis and enhancing hippocampal synaptic plasticity.
Although our study demonstrates that intravenous transplantation of hUC-MSCs
significantly improves cognitive function, the mechanistic basis remains unclear. In
the further study, we will investigate the mechanism of hUC-MSCs on the activation
of endogenous neurogenesis and the reconstruction of synaptic connectivity mediated
by Sirt1 and BDNF.
Taken together, our findings indicate that intravenous transplantation of
hUC-MSCs improves cognitive function in a 12-month-old Tg2576 mouse model that
exhibits well-established Aβ deposition by promoting neurogenesis and synaptic
plasticity, increasing protein levels of Sirt1, BDNF and SYN and decreasing
hippocampal oxidative stress. Our results also suggest that modulating hUC-MSCs to
generate excess neuroprotective factors could provide a viable therapy to treat AD.

Authors’ contributions

YBC designed and performed the experiments and wrote the manuscript. SSM, FXG
and BY participating in designing and guiding the experiments. CYZ, WC, ML and
QX provided assistance for ELISA assay, hUC-MSCs, infusion data analysis and
MWM test respectively. RNQ, NY, PJL and DPL were responsible for
immunochemistry and immunofluorescence test. All authors read and approved the
manuscript for publication.
Funding: This work were supported by the Natural Science Foundation of China
(NSFC 81471306, U1404313). The Innovative Research Team in Science and
Technology of the University of Henan Province (15IRTSTHN022). The Plan for
Scientific Innovation Talent of Henan Province (154200510008). The International
Collaboration Research Program for Talents of Henan Province (2016GH15,
2016GH03). The Key Research Project of Higher Education of Henan Province
(17A310012). The Research Fund for the Doctoral Program of Higher Education of
China (20114101110004).

Disclosure statement

The authors have no competing interests.

Acknowledgements

This work were supported by the Natural Science Foundation of China (NSFC
81471306, U1404313). The Innovative Research Team in Science and Technology of
the University of Henan Province (15IRTSTHN022). The Plan for Scientific
Innovation Talent of Henan Province (154200510008) and The International
Collaboration Research Program for Talents of Henan Province (2016GH15,
2016GH03). The Key Research Project of Higher Education of Henan Province
(17A310012). The Research Fund for the Doctoral Program of Higher Education of
China (20114101110004). We thank Dr. Derek Huffman for his suggestions and
revision on the manuscript and all the team members in Dr. Guan’s lab for their
discussions and assistance for the project.
References

Querfurth HW, LaFerla FM,2010. Alzheimer’s disease. N Engl J Med.362: 329–344.

Thies W, Bleiler L,2011.Alzheimer’s disease facts and figures. Alzheimers Dement.2011:208–244.

Selkoe DJ,2001. Alzheimer’s disease: Genes, proteins, and therapy. Physiol Rev.81:741–766.

Cummings JL, Morstorf T, Zhong K,2014. Alzheimer’s disease drug-development pipeline: few

candidates, frequent failures. Alzheimers Res Ther. 6: 37.

Holtzman DM, John CM, Goate A,2011. Alzheimer’s Disease: The Challenge of the Second

Century. Sci Transl Med.3(77):1-35.

Miller G. Pharmacology,2010. The puzzling rise and fall of a dark-horse Alzheimer’s drug.

Science.327: 1309.

Alzheimer's Association,2010. Alzheimer’s disease facts and figures. Alzheimers

Demen.6(2):158–194.

Blurton-Jones M, Kitazawa M, Martinez-Coria H,2009. Neural stem cells improve cognition via

BDNF in a transgenic model of Alzheimer disease. Pro Natl Acad Sci.106(32): 13594-13599.

Lee I-Shin, Jung Kwangsoo, Kim I-Sun, Lee Haejin, Kim Miri, Yun Seokhwan, Hwang Kyujin,

Shin Jeong Eun, Park Kook In,2015. Human neural stem cells alleviate Alzheimer like

pathology in a mouse model. Mol Neurodegener.10:38-46.

Yan Y, Ma T, Gong K,2014. Adipose-derived mesenchymal stem cell transplantation promotes

adult neurogenesis in the brains of Alzheimer's disease mice. Neural Regen Res.9(8):

798-806.

Babaei P, Soltani Tehrani B, Alizadeh A,2012. Transplanted bone marrow mesenchymal stem cells

improve memory in rat models of Alzheimer's disease. Stem Cells Int. 20(1):156-164.

Ma T, Gong K, Ao Q,2013. Intracerebral transplantation of adipose-derived mesenchymal stem

cells alternatively activates microglia and ameliorates neuropathological deficits in

Alzheimer’s disease mice. Cell Transplant.22(1): 113-126.

Yang H, Xie Z, Wei L,2013. Human umbilical cord mesenchymal stem cell-derived neuron-like

cells rescue memory deficits and reduce amyloid-beta deposition in an AβPP/PS1 transgenic

mouse model. Stem Cell Res Ther. 4(4): 76-82.

Shih-Yi Kao,Jia-Fwu Shyu,Hwai-Shi Wang,Chi-Hung Lin,Cheng-Hsi Su,Tien-Hua

Chen,Zen-Chung Weng,Pei-Jiun Tsai,Jerry Chan,2015. Comparisons of Differentiation

Potential in Human Mesenchymal Stem Cells from Wharton’s Jelly, Bone Marrow, and

Pancreatic Tissues. Stem Cells Int.2015:232-241.

Zhou Changhui,Yang Bo,Tian Yi,Jiao Hongliang,Zheng Wendi,Wang Jian,Guan Fangxia,2011.

Immunomodulatory effect of human umbilical cord Wharton's jelly-derived mesenchymal

stem cells on lymphocytes. Cell Immunol. 272(1): 33–38.

Toupadakis Chrisoula A,Wong Alice,Genetos Damian C,Cheung Whitney K,Borjesson Dori

L,Ferraro Gregory L,Galuppo Lawrence D,Leach J Kent,Owens Sean D,Yellowley Clare

E,2010. Comparison of the osteogenic potential of equine mesenchymal stem cells from bone

marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue. Am J Vet

Res.71(10):1237-1245.

Kim Saeromi,Chang Keun-A,Kim Jeong a,Park Hyeong-Geun,Ra Jeong Chan,Kim Hye-Sun,Suh

Yoo-Hun,2012. The preventive and therapeutic effects of intravenous human adipose-derived

stem cells in Alzheimer's disease mice. PLoS One.7(9): e45757.

Zhang W, Wang P J, Sha H,2014. Neural stem cell transplants improve cognitive function without

altering amyloid pathology in an APP/PS1 double transgenic model of Alzheimer’s disease.

Mol Neurobiol.50(2): 423-437.

Ehrhart J, Darlington D, Kuzmin-Nichols N,2016. Biodistribution of Infused Human Umbilical

Cord Blood Cells in Alzheimer’s Disease-Like Murine Model. Cell Transplant.25(1):

195-199.

Kim KS, Kim HS, Park JM, Kim HW, Park MK, Lee HS, Lim DS, Lee TH, Chopp M, Moon

J,2013. Long-term immunomodulatory effect of amniotic stem cells in an Alzheimer's disease

model. Neurobiol Aging. 34(10):2408-2420.

Park S E, Lee N K, Lee J,2016. Distribution of human umbilical cord blood-derived mesenchymal

stem cells in the Alzheimer's disease transgenic mouse after a single intravenous injection.

Neuroreport.12(8):112-118.

Xie ZH, Liu Z, Zhang XR, Yang H, Wei LF, Wang Y, Xu SL, Sun L, Lai C, Bi JZ, Wang XY,2016.

Wharton's Jelly-derived mesenchymal stem cells alleviate memory deficits and reduce

amyloid-β deposition in an APP/PS1 transgenic mouse model. Clin Exp Med.16(1):89-98.

Zohreh Bagher,Somayeh Ebrahimi-Barough,Mahmoud Azami,Hamid Mirzadeh,Mansooreh

Soleimani,Jafar Ai,Mohammad Reza Nourani,Mohammad Taghi Joghataei,2015. Induction

of human umbilical Wharton's jelly-derived mesenchymal stem cells toward motor

neuron-like cells. In Vitro Cell Dev Biol Anim.51(9):987-994.

Ruzicka J,Kulijewicz-Nawrot M,Rodrigez-Arellano JJ,Jendelova P,Sykova E,2016. Mesenchymal

Stem Cells Preserve Working Memory in the 3xTg-AD Mouse Model of Alzheimer's

Disease.Int J Mol Sci. 17(2):152.

Liu L,Yu Y,Hou Y,Chai J,Duan H,Chu W,Zhang H,Hu Q,Du J,2014. Human umbilical cord

mesenchymal stem cells transplantation promotes cutaneous wound healing of severe burned

rats. PLoS One. 9(2):e88348.

Chen SQ,Cai Q,Shen YY,Wang PY,Li MH,Teng GY,2014. Neural stem cell transplantation

improves spatial learning and memory via neuronal regeneration in amyloid-β precursor

protein/presenilin 1/tau triple transgenic mice. Am J Alzheimers Dis Other Demen.

29(2):142-9.

Yue W, Li Y,Zhang T,Jiang M,Qian Y,Zhang M,Sheng N,Feng S,Tang K,Yu X,Shu Y,Yue

C,JingN,2015.ESC-Derived Basal Forebrain Cholinergic Neurons Ameliorate the Cognitive

Symptoms Associated with Alzheimer's Disease in Mouse Models. Stem Cell Reports.

5(5):776-90.

Hunsberger J G, Rao M, Kurtzberg J,2016. Accelerating stem cell trials for Alzheimer's disease.

Lancet Neurol.15(2): 135-138.

Khabbal J, Kerkela E, Mitkari B, Raki M, Nystedt J, Mikkonen V,2015. Differential Clearance of

Rat and Human Bone Marrow-Derived Mesenchymal Stem Cells From the Brain After

Intra-arterial Infusion inRats. Cell Transplant. 24(5):819–28.

Kim JA,Ha S,Shin KY,Kim S,Lee KJ,Chong YH,Chang KA,Suh YH,2015. Neural stem cell

transplantation at critical period improves learning and memory through restoring synaptic

impairment in Alzheimer's disease mouse model. Cell Death Dis. 18;6:e1789.

Xuan AG, Luo M, Ji WD, Long DH,2009. Effects of engrafted neural stem cells in Alzheimer’s

disease rats. Neurosci Lett.450:167–171.

Sultana Rukhsana, Butterfield D Allan,2010. Role of oxidative stress in the progression of

Alzheimer's disease. J Alzheimers Dis.19(1):341-353.

A. Tramutola, C. Lanzillotta,M. Perluigi,D. Allan Butterfield,2016. Oxidative Stress, Protein

Modification and Alzheimer Disease. Brain Res Bull.16:30129-30140.

Chiang MC,Nicol CJ,Cheng YC,Lin KH,Yen CH,Lin CH,2016. Rosiglitazone activation of

PPARγ-dependent pathways is neuroprotective in human neural stem cells against

amyloid-beta-induced mitochondrial dysfunction and oxidative stress. Neurobiol Aging.

40:181-90.

Chakroborty S,Kim J,Schneider C,West AR,Stutzmann GE,2015. Nitric oxide signaling is

recruited as a compensatory mechanism for sustaining synaptic plasticity in Alzheimer's

disease mice. J Neurosci. 35(17):6893-902.

Kwon KJ,Park JH,Jo I,Song KH,Han JS,Park SH,Han SH,Cho DH, 2016. Disruption of neuronal

nitric oxide synthase dimerization contributes to the development of Alzheimer's disease:

Involvement of cyclin-dependent kinase 5-mediated phosphorylation of neuronal nitric oxide

synthase at Ser293. Neurochem Int. 99:52-61.

Michán Shaday,Li Ying,Chou Maggie Meng-Hsiu,Parrella Edoardo,Ge Huanying,Long Jeffrey

M,Allard Joanne S,Lewis Kaitlyn,Miller Marshall,Xu Wei,Mervis Ronald F,Chen

Jing,Guerin Karen I,Smith Lois E H,McBurney Michael W,Sinclair David A,Baudry

Michel,de Cabo Rafael,Longo Valter D,2010. SIRT1 is essential for normal cognitive

function and synaptic plasticity. J Neurosci.30(29): 9695-707.

Cho SH,Chen JA,Sayed F,Ward ME,Gao F,Nguyen TA,Krabbe G,Sohn PD,Lo I,Minami

S,Devidze N,Zhou Y,Coppola G,Gan L,2015. SIRT1 deficiency in microglia contributes to

cognitive decline in aging and neurodegeneration via epigenetic regulation of IL-1β.J

Neurosci. 35(2):807-18.

Gottschalk W A,Jiang H,Tartaglia N,Feng L,Figurov A,Lu B,1999. Signaling mechanisms

mediating BDNF modulation of synaptic plasticity in the hippocampus. Learn

Mem.6(3):245-256.

Abigail Mariga,Jiri Zavadil,Stephen D. Ginsberg,Moses V. Chao,2015. Withdrawal of BDNF from

hippocampal cultures leads to changes in genes involved in synaptic function. Devel

Neurobio.75(2):173-192.

Janna Aarse,Stefan Herlitze,Denise Manahan-Vaughan,2016. The requirement of BDNF for

hippocampal synaptic plasticity is experience-dependent. Hippocampus.26(6):739-751.

Tanja Novkovic,Thomas Mittmann,Denise Manahan-Vaughan,2015. BDNF contributes to the

facilitation of hippocampal synaptic plasticity and learning enabled by environmental

enrichment. Hippocampus.25(1):1-15.

Loredana Zocchi,Paolo Sassone-Corsi,2012. SIRT1-mediated deacetylation of MeCP2 contributes

to BDNF expression. Epigenetics.7(7):695-700.

Kwon Sung E,Chapman Edwin R,2011. Synaptophysin regulates the kinetics of synaptic vesicle

endocytosis in central neurons. Neuron.70(5):847-854.

Tampellini Davide,Capetillo-Zarate Estibaliz,Dumont Magali,Huang Zhenyong,Yu Fangmin,Lin

Michael T,Gouras Gunnar K,2010. Effects of synaptic modulation on beta-amyloid,

synaptophysin, and memory performance in Alzheimer's disease transgenic mice. J

Neurosci.30(43): 14299-304.

Ming Li, Kequan Guo, Luca Vanella, Shigeru Taketani,Yasushi Adachi, Susumu Ikehara,2015.

Stem Cell Transplantation Upregulates Sirt1 and Antioxidant Expression, Ameliorating Fatty

Liver in Type 2 Diabetic Mice. Int J Biol Sci. 11(4): 472–481.

Shanshan Ma,Shuo Liang,Hongliang Jiao,Liankai Chi,Xinyi Shi,Yi Tian,Bo Yang,Fangxia

Guan,2014. Human umbilical cord mesenchymal stem cells inhibit C6 glioma growth via

secretion of dickkopf-1 (DKK1). Mol Cell Biochem.385(1-2):277-286.

Li H, Liang A, Guan F, Fan R, Chi L, Yang B,2013. Regular treadmill running improves spatial

learning and memory performance in young mice through increased hippocampal

neurogenesis and decreased stress. Brain Res. 1531:1-8.

Rahasson R. Ager, Joy L. Davis, Andy Agazaryan, Francisca Benavente, Wayne W.Poon, Frank M.

LaFerla, and Mathew Blurton-Jones,2015. Human Neural Stem Cells Improve Cognition and

Promote Synaptic Growth in Two Complementary Transgenic Models of Alzheimer’s

Disease and Neuronal Loss. Hippocampus. 25(7): 813–826.

Yang H, Yue C, Yang H, Xie Z, Hu H, Wei L, Wang P, Zhao C, Bi J,2013.Intravenous

Administration of Human Umbilical Cord Mesenchymal Stem Cells Improves Cognitive

Impairments and Reduces Amyloid-Beta Deposition in an AβPP/PS1 Transgenic Mouse

Model. Neurochem Res. 38(12):2474-2482.