Hindawi

BioMed Research International

Volume 2020, Article ID 2863501, 13 pages
https://doi.org/10.1155/2020/2863501

Research Article
Multiple Intravenous Injections of Valproic Acid-Induced
Mesenchymal Stem Cell from Human-Induced Pluripotent Stem
Cells Improved Cardiac Function in an Acute Myocardial
Infarction Rat Model

Shuyuan Guo,1 Yusen Zhang,2 Yanmin Zhang,3 Fanhua Meng,4 Minghua Li,3 Zhendong Yu,3
Yun Chen ,2,5 and Guanghui Cui 2
1
Shantou University, School of Medicine, China
2
Peking University Shenzhen Hospital, Ultrasound Department, China
3
Peking University Shenzhen Hospital, Central Laboratory, China
4
Peking University Shenzhen Hospital, Reproductive Medical Center, China
5
Shenzhen Key Laboratory of Drug Addiction and Safe Medication, China

Correspondence should be addressed to Yun Chen; yunchen@sphmc.org and Guanghui Cui; cuiguanghui175@aliyun.com

Received 21 April 2020; Revised 10 November 2020; Accepted 2 December 2020; Published 17 December 2020

Academic Editor: Ruxana Sadikot

Copyright © 2020 Shuyuan 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.

Mounting evidence indicates that the mesenchymal stem cell (MSC) injection is safe and efficacious for treating cardiomyopathy;
however, there is limited information relating to multiple intravenous injections of human-induced pluripotent stem cell-derived
mesenchymal stem cell (hiPSC-MSC) and long-term evaluation of the cardiac function. In the current study, MSC-like cells were
derived from human-induced pluripotent stem cells through valproic acid (VPA) induction and continuous cell passages. The
derived spindle-like cells expressed MSC-related markers, secreted angiogenic and immune-regulatory factors, and could be
induced to experience chondrogenic and adipogenic differentiation. During the induction process, expression of epithelial-to-
mesenchymal transition- (EMT-) related gene N-cadherin and vimentin was upregulated to a very high level, and the expression
of pluripotency-related genes Sox2 and Oct4 was downregulated or remained unchanged, indicating that VPA initiated EMT by
upregulating the expression of EMT promoting genes and downregulating that of pluripotency-related genes. Two and four
intravenous hiPSC-MSC injections (106 cells/per injections) were provided, respectively, to model rats one week after acute
myocardial infarction (AMI). Cardiac function parameters were dynamically monitored during a 12-week period. Two and four
cell injections significantly the improved left ventricular ejection fraction and left ventricular fractional shortening; four-
injection markedly stimulated angiogenesis reduced the scar size and cell apoptosis number in the scar area in comparison with
that of the untreated control model rats. Although the difference was insignificant, the hiPSC-MSC administration delayed the
increase of left ventricular end-diastolic dimension to different extents compared with that of the PBS-injection control. No
perceptible immune reaction symptom or hiPSC-MSC-induced tumour formation was found over 12 weeks. Compared with the
PBS-injection control, four injections produced better outcome than two injections; as a result, at least four rounds of MSC
injections were suggested for AMI treatment.

1. Introduction damage to cardiomyocytes after AMI leads to left ventricular

(LV) remodeling and ischemic heart failure [1, 2].
Coronary artery disease is a major cause of morbidity and Mesenchymal stem cells (MSCs) are mesoderm-derived
mortality worldwide. Despite recent achievements in acute cells with the capacity to differentiate into multicell lineages.
myocardial infarction (AMI) treatment, the irreversible MSCs exert an immune tolerant phenotype which is
2 BioMed Research International

Table 1: Primers used in quantitative real-time RT-PCR.

Genes PCR primers

Sense primer:5 ′ -TTCTGCTGCTCTTGCTGTTT-3 ′
E-cadherin
Antisense primer: 5 ′ -TGGCTCAAGTCAAAGTCCTG-3 ′
Sense primer: 5 ′ -CCTGCGCGTGAAGGTTTGCC-3 ′
N-cadherin
Antisense primer: 5 ′ -CCAAGCCCCGCACCCACAA-3 ′
Sense primer: 5 ′ -GCCCTTAAAGGAACCAATGA-3 ′
Vimentin
Antisense primer: 5 ′ -AGCTTCAACGGCAAAGTTCT-3 ′
Sense primer: 5 ′ -GACAGGGGGAGGGGAGGAGCTAGG-3 ′
Oct3/4
Antisense primer: 5 ′ -CTTCCCTCCAACCAGTTGCCCCAAAC-3 ′
Sense primer: 5 ′ -GGGAAATGGGAGGGGTGCAAAAGAGG-3 ′
Sox2
Antisense primer: 5 ′ -TTGCGTGAGTGTGGATGGGATTGGTG-3 ′
Sense primer: 5 ′ -CATCAATGGAAATCCCATCA-3 ′
GAPDH
Antisense primer: 5 ′ -TTCTCCATGGTGGTGAAGAC-3 ′

characterized by low level expression of MHC antigens and of MSCs [21]. Completely curbing excessive and prolonged
the lack of costimulatory molecules. Through releasing solu- inflammation in myocardium by a single intravenous admin-
ble immune modulators such as indoleamine 2, 3 dioxygen- istration of MSCs is impossible; therefore, repeated infusions
ase (IDO), prostaglandin E2 (PGE-2), or nitric oxide (NO), over time might exert superior, prolonged anti-inflammatory
MSCs suppress activation of immune cells [3]. effects.
In recent years, mesenchymal stem cell transplantation Despite the advantages of using MSCs derived from the
has emerged as a potential treatment means for ischemic bone marrow, adipose tissue, or umbilical cord for cell ther-
heart disease [4–6]. In clinical trials, intracoronary delivery apy, several issues incurred by properties of these cells limit
of bone marrow mononuclear cells or bone marrow MSCs their application in clinical use. For example, they are limited
has provided evidence of improved cardiac function, reduced in number and quickly lose their differentiation potential
scar area, and reduced prevalence of recurrent MI, stent during in vitro expansion [22]. As for autologous application,
thrombosis, and death [7–11]. Accumulating evidence has MSCs decline in quantity and quality with age, isolated MSCs
suggested that following AMI, chronic, excessive proinflam- are often composed of heterogeneous cell populations, and
matory immune responses may account for progressive each population may have different proliferative and differ-
adverse remodeling and dysfunction of the myocardium, entiation potential that make clinic application difficult [23,
and the transplanted MSCs exert its therapeutic benefits by 24]. A large number of primitive MSCs can be isolated from
secreting factors to stimulate local angiogenesis and alleviate the perinatal tissue, but it can be obtained from only around
inflammatory activities [12–15]. one-third of umbilical cord blood specimens, and the access
To date, most experimental and clinical studies have used to the abortal tissue requires ethical approval [25, 26].
an intramyocardium or intracoronary delivery system. Intra- The human induced pluripotent stem cell (hiPSC) is an
venous cell delivery is advantageous for AMI treatment at a attractive stem cell source for cell therapy. In comparison to
practical level, and multiple rounds of intravenous injections isolation of primary cells, iPSCs can be harvested free of eth-
of MSCs are convenient and tolerable from the viewpoint of ical constraints and offer a unified starting point for genera-
patients, whereas repeated intromyocardium or introcoronary tion of standardised derivatives at large scale [27]. Thus,
cell delivery is not. However, numerous reports indicate that differentiating hiPSC into MSC (hiPSC-MSC) before trans-
only a small portion of the injected cells could integrate into plantation is one of the most promising approaches for the
the infarct area after intravenous injections, while most of safe and effective use of the induced pluripotent stem cells.
them are trapped in the lung, kidney, liver, and spleen, and Before clinic application of the induced hiPSC-MSC,
are gradually cleared away in a period of time [16–18]. Even there are questions remaining to be answered. First, do the
with the application of molecule targeting delivery systems, intravenous injected hiPSC-MSCs have tumor formation
such as ICAM or SDF1/CXCR4, the retaining rate of the potential in the hosts? Second, will immune reaction to
injected cells in the infracted area is still less than 30% [19, 20]. transplanted cells be initiated and accelerated by repeated
Ne´ meth et al. demonstrated that the exogenous MSCs injections of hiPSC-MSCs? Third, what is the optimal cell
accumulated in the lung could transmigrate outside to the vas- dosage for improving the cardiac function without causing
cular space and rapidly interact with lung-resident tissue mac- fatal side effects?
rophages; stimulated by the MSCs, endogenous macrophages Valproic acid (VPA), a histone deacetylase inhibitor
produce large amount of interleukin-10. Since this seminal (HDI), is used as a mood stabiliser and antiepileptic drug.
report, there has been a plurality of reports validating a unique Recently, it has been found that VPA treatment could mod-
cross-talk between exogenous MSCs and recipient monocyte ulate multipotency, migration, proliferation, and differentia-
and/or macrophages as part of the anti-inflammatory effect tion of cord blood and periodontal MSCs [28, 29].
BioMed Research International 3

(a) (b)

(c) (d)

(e) (f)

Figure 1: Morphology transformation during hiPSC-MSC induction. (a) Undifferentiated human iPSC colony. (b) hiPSCs transformed to
larger cuboidal-shaped epithelial-like cells after eight-day VPA induction. (c) After the first passage, the morphology of differentiating cells
transformed from cuboidal to olive shape. (d) Large spindle-shaped cells appeared in the second passage. (e) Small spindle-shaped cells grew
out from cell mess. (f) On the passage six, more than 90% of the adherent cells maintained a small spindle-shaped morphology 100×.

Epithelial-to-mesenchymal transition (EMT) has been of AMI. By doing so, we attempted to test the safety and
regarded as the first stage of mesoderm commitment in the effectiveness of the derived MSC-like cells in treating
hESCs [30], and VPA plays an important role in the process AMI within a 12-week period.
of cell mesoderm commitment induction [31].
In this study, we hypothesised that MSC-like cells could 2. Materials and Methods
be derived from human hiPSCs by VPA induction and
planned to undertake two and four rounds of intravenous 2.1. Human-Induced Pluripotent Stem Cell Cultures. Human
administration of the derived hiPSC-MSCs in a rat model iPSC cell line PBMC-5 (derived from the peripheral blood
4 BioMed Research International

mononuclear cell of a 31-year-old Chinese Han female) was 14

kindly provided by Dr Tiancheng Zhou at the Guangzhou
Institute of Biomedicine & Health, Chinese Academy of Sci- 12
ences, under the appropriate human ethics and material

Relative gene expression level

transfer agreements. hiPSCs were routinely grown as bulk 10
cultures on a widely used commercially available defined
medium, mTeSR1 (Stem Cell Technologies, Vancouver, BC, 8
Canada), in combination with the cell attachment matrix
Matrigel (BD Biosciences, San Diego, USA). 6

2.2. MSC Differentiation of hiPSC. hiPSC bulk cultures were 4

seeded into the mTeSR1 medium on a Matrigel-coated six-
well plate as large colonies at high confluence, and then the 2
medium was changed to the MSC differentiation medium
containing DMEM-Ham’s F-12 basal medium supplemented 0
N-Cadherin Vimentin E-Cadherin Oct4 Sox2
with 20% knockout serum replacement (KOSR), 1 mM L-
glutamine, 10 mM nonessential amino acids, 50 U/mL peni- 4 days
cillin/streptomycin (Invitrogen, Carlsbad, CA, USA), and 8 days
0.5 mM VPA (Sigma-Aldrich, St. Louis, USA). The MSC dif-
Figure 2: VPA treatment up-regulated EMT-related gene
ferentiation medium was replaced every other day for eight expression.
days, and then the cells were passaged to a single cell suspen-
sion using the TrypleSelect (Invitrogen). Single cells were staining of induced cells were conducted as described previ-
seeded at a density of 40,000 cells per cm2 into the MSC ously [32]. Chondrogenic differentiation was induced by cul-
medium (DMEM high glucose basal medium supplemented turing cells with a chondrogenic induction medium (DMEM
with 10% foetal bovine serum, 1 mM L glutamine, 50 U/mL supplemented with 10% FBS, 6.25 μg/mL of insulin, 50 nM of
penicillin/streptomycin, and 10 mM nonessential amino ascorbate phosphate, and 10 ng/mL of transforming growth
acids, all from Invitrogen) as the first passage, and then in factor-β) for two weeks. Formation of sulphated proteogly-
the subsequent passages, 20,000 cells per cm2 were seeded. cans was visualised by Alcian Blue staining.
To induce adipocytic differentiation, cells were cultured
2.3. Quantitative Real-Time RT-PCR. Total RNA was isolated
as monolayers, allowing to become near confluent and then
using the RNase mini kit (Qiagen, Valencia, CA, USA). A
maintained in the adipogenic induction medium (DMEM
high-capacity RNA-to-cDNA kit (Applied Biosystems, Fos-
supplemented with 10% FBS, 10−7 M of dexamethasone,
ter City, CA, USA) was used to reverse transcribe 1 μg of
and 10 μg/mL of insulin) for two weeks. Then, cells were
RNA into cDNA with MMLV reverse transcriptase and ran-
assessed by Oil-Red-O staining as an indicator of intracellu-
dom primers according to the manufacturer’s instructions
lar lipid accumulation.
(85°C, 5 s, and 37°C, 1 h). Real-time PCR was performed
using specific primers under optimal reaction conditions
(94°C, 30 s; 60°C, 35 s; 72°C, 40 s; 30 cycles) for quantification 2.6. Cytokine Production Analysis. Two million umbilical
of the gene expression using SYBR-Green RT-PCR reagents cord derived MSCs (UC-MSCs) and hiPSC-MSCs were,
(Applied Biosystems). The 2-ΔΔCq method was used to respectively, seeded in a 10 cm diameter culture dish in the
analyze relative gene expression level. Table 1 lists the MSC medium for the purpose of comparing the cytokine
primers used. production level between these two cell groups. After a
three-day culture period, the supernatants were collected
2.4. Detection of MSC Specific Markers by Flow Cytometry. for detecting IGF, VEGF, HGF, IDO, PGE2, and IL-10 pro-
Adherent spindle-like cells at the sixth passage were har- duction. Cytokine concentration was measured using ELISA
vested by trypsinization. They were resuspended for measur- detection kits according to the manufacturer’s instructions
ing expression of typical MSC surface markers (CD29, CD44, (IGF and PGE2, Multi Science, Hangzhou, China; VEGF
CD73, CD90, and CD105) as well as markers of hematopoi- and HGF, Abcam, Cambridge, MA, USA; IDO, RayBio Nor-
etic cell lineages (CD34, CD45, and human leukocyte cross, GA, USA; IL-10, Sino Biological, Beijing, China). Sam-
antigen-DR isotype) by fluorescent-activated cell sorting ples were analyzed using a 1510 spectrophotometer (Thermo
(FACS). Fluorescent conjugated antibodies: anti-CD29, Fisher, Vantaa, Finland).
CD73, CD90, and CD105 (Biolegend, San Diego, CA,
USA); anti-CD45, CD34, CD44 and HLA-DR (Boster,
Wuhan, China). Isotype control antibodies were used as neg- 2.7. Animals and AMI Induction. Male Sprague–Dawley rats
ative controls to optimise parameters during analyses using (4 weeks, body mass 230-270 g) were purchased from the
BD-FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). Medical Experimental Animal Centre of Guangdong Prov-
Data were analyzed using FlowJo (Ashland, OR, USA). ince (Guangdong, China). They were housed in a room at
constant temperature (20°C) and humidity (45%) on a 12-h
2.5. Differentiation Assay of hiPSC-MSC. Induction of chon- light–dark cycle. Rats had free access to standard laboratory
drogenic and adipogenic differentiation and subsequent chow and tap water.
BioMed Research International 5

(a) (b)

(c) (d)

Figure 3: hiPSC-MSC chondrogenic and adipogenic differentiation induction. (a) Alcian Blue staining for sulphated proteoglycans. (b)
Negative control. (c) Oil-Red-O staining for intercellular lipid vacuoles. (d) Negative control 400×.

Approval for animal experimentation was obtained from echocardiography system (Vevo 2100; Visual Sonics,
the Ethics Committee of Peking University Shenzhen Hospi- Toronto, ON, Canada) with an MS250 transducer (13–
tal. Chinese laws relating to the care and treatment of animals 24 MHz). We measured parameter changes pertaining to
were followed strictly throughout the study and complied the left ventricular ejection fraction (LVEF), left ventricular
with Animal Research: Reporting of In Vivo Experiments fractional shortening (LVFS), and left ventricular end-
(ARRIVE) guidelines. diastolic dimension (LVEDD).
A left thoracotomy was undertaken between the fourth
and fifth ribs of rats. A suture was constructed around the left 2.9. Intravenous Administration of hiPSC-MSC in the Rat
anterior descending coronary artery (LADCA). Ischemia was Model of AMI. One week after AMI, model rats were anaes-
maintained for 40 min, followed by reperfusion. In sham thetised (40 mg/kg body mass of pentobarbital sodium),
control rats, the constructed suture was immediately and the cardiac function was evaluated by echocardiography.
removed to restore blood flow. Rats with LVEF lower than 55% were selected for this exper-
iment and divided randomly into two hiPSC-MSC injection
2.8. Evaluation of the Cardiac Function by Echocardiography. groups (two and four injections, n = 8) and one phosphate-
The cardiac function was evaluated using a two-dimensional buffered saline (PBS) injection control group (n = 8). Two
6 BioMed Research International

CD 29 CD 44 CD 73 CD 90
0.50 99.5 1.30 98.7 4.0K 0.20 99.8 0.60 99.4
3.0K
2.0K 1.5K
3.0K
2.0K 1.5K

Count
Count

Count
Count
1.0K
2.0K
1.0K
1.0K 500K
1.0K
500

0 0 0 0
100 102 104 106 108 101 103 105 107 10–3 100 103 106 109 100 102 104 106
CD 105 CD 34 CD 45 HLA-DR
0.80 99.2 3.0K 99.6 0.40 1.2K 99.9 0.10 99.8 0.20
2.5K 1.5K

2.0K 900
2.0K 1.0K
Count

Count

Count
Count

1.5K
600
1.0K
1.0K 500
300
500

0 0 0 0
100 102 104 106 101 103 105 107 100 102 104 106 108 1010 100 102 104 106

Figure 4: Surface marker expression detection of hiPSC-MSC.

3
and four intravenous boli of 1 × 106 hiPSC-MSCs (passage 6)
were given, respectively, to rats in the hiPSC-MSC-treated
groups, and PBS was provided to rats in the control group. 2.5
hiPSC-MSC intravenous injections were carried out once a
week. The cardiac function was evaluated once every two 2
OD 450 nm

weeks after the first infusion in the 12-week period.

1.5
2.10. Quantification of the Scar Size. Masson’s Trichrome ⁎⁎⁎
staining was used to assess the area of myocardial scars 12 1
weeks after AMI induction. This procedure stains the viable
area pink and the scar area blue. Briefly, three equal slices 0.5
below the suture were fixed in 4% formaldehyde and embed-
ded in paraffin and cut into sections of thickness 5 μm. To
0
detect fibrosis, the sections were stained with Masson’s VEGF IGF HGF IDO PGE2 IL-10
Trichrome and assessed by a pathologist blind as to study
protocol. The percentage of the scar area was quantified by UC-MSC
a computerised morphometry system with a microscope VPA-MSC
(Olympus, Tokyo, Japan).
Figure 5: hiPSC-MSC produced angiogenic and immune regulatory
factors.
2.11. Vascularisation Evaluation in the Scar Area. Three fro-
zen equal slices below the suture were fixed and blocked for
immunohistochemical detection of the α-SMA expression. cyte apoptosis. Counterstaining with hematoxylin was con-
Primary mouse anti-rat α-SMA antibodies (Biolegend, San ducted to visualize normal nuclei. Apoptotic cells were
Diego, CA, USA), secondary biotin-coupled anti-mouse counted in 5 randomly selected high-power fields (400×)
IgG antibody, and avidin-horseradish peroxidase conjugates from the border and infarcted area of three sections from
(Boster, Wuhan, Hubei, China) were applied, respectively, each heart and averaged.
following a standard immunohistochemistry procedure.
Irrelevant antibody was used as a negative control. α-SMA+ 2.13. Statistical Analyses. Data were shown as mean ± SD and
cell capillary in the scar regions was counted by a high- analyzed using SPSS software, Version 11.0. One-way
power field (200×) in each section, and at least five randomly ANOVA with LSD- t was used in multiple group compari-
fields in each section were analyzed. sons, and P < 0:05 was considered significant.

2.12. Cell Apoptosis Assay. The TdT-mediated dUTP nick- 3. Results

end labeling (TUNEL) assay was performed on heart sections
according to the manufacturer’s recommendations (KeyGEN 3.1. Morphology Transformation during hiPSC-MSC
BioTECH, Nanjing, Jiangsu, China) to quantify cardiomyo- Induction. Without the presence of VPA, undifferentiated
BioMed Research International 7

90
60
80 ⁎
70 50
80 ⁎⁎⁎
60 ⁎⁎⁎
40
LVEF (%)

LVFS (%)
50 60
30

LVEF (%)
40
40
30 20
20
20 10
10
0 0 0
Week Week Week Week Week Week Week 4-injection 2-injection PBS-injection Week Week Week Week Week Week Week
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Sham group 2-injection Sham group 2-injection
4-injection PBS-injection 4-injection PBS-injection

(a) (b) (c)

12

10
⁎⁎⁎ 12
50
8
⁎⁎⁎
LVEDD (mm)

11
40

LVEDD (mm)
6
10
LVFS (%)

30
4
20 9

10 2 8

0 0 7
4-injection 2-injection PBS-injection Week Week Week Week Week Week Week 4-injection 2-injection PBS-injection
0 2 4 6 8 10 12
Sham group 2-injection
4-injection PBS-injection

(d) (e) (f)

Figure 6: hiPSC-MSC administration improved the cardiac function. (a) LVEF of the PBS-injection group continued to decline from week 0
to week 12. hiPSC-MSC injection increased LVEF to different extents in cell-treated groups. (b) The improvement in LVEF in the hiPSC-
MSC-injection groups was significant compared with that in the PBS-injection group at week 12 (PBS-injection group v. two-injection
group, ∗∗∗ P < 0:001; PBS-injection group v. four-injection group, ∗∗∗ P < 0:001). There was a significant difference between the two-
injection and the four-injection groups, ∗ P < 0:05. (c) LVFS of the PBS-injection group kept declining from week 0 to week 12. hiPSC-
MSC treatments increased LVFS to different levels in the MSC-injection groups. (d) Significant differences were found when comparing
the improvement extent of LVFS of the hiPSC-MSC-injection groups with that of the PBS-injection group at the end of week 12 (PBS-
injection group v. two-injection group, ∗∗∗ P < 0:001; PBS-injection group v. four-injection group, ∗∗∗ P < 0:001). (e) In the PBS-injection
group, LVEDD increased continuously over 12 weeks. hiPSC-MSC treatments prevented the increase of LVEDD in the hiPSC-MSC-
injection groups to different levels compared with that of the PBS-injection group. (f) The differences of LVEDD prevention among the
hiPSC-MSC-injection groups and the PBS-injection group were insignificant at the end of 12 weeks.

hiPSCs cultured on Matrigel in the mTeSR1 medium derm commitment, pluripotency-related transcription fac-
appeared as dense, tightly packed cell colonies. Eight-day tors and EMT-related gene expression were evaluated at the
incubation with VPA drove more than 60% of hiPSCs to fourth and eighth induction days. In comparison with
form a monolayer of larger cuboidal-shaped epithelial-like DMSO-treated control hiPSCs, four-day VPA induction sig-
cells, leaving only small islands of undifferentiated cells. nificantly upregulated the expression of the EMT-related
From the second passage in the MSC medium, the morphol- gene N-cadherin and vimentin, and the upregulation was
ogy of differentiating cells gradually altered from larger continued to day eight. The pluripotency-related transcrip-
cuboidal-shaped epithelial-like towards olive shape-like, tion factor Sox2 was downregulated, while the Oct4 expres-
and then on the sixth passage, more than 90% of the adherent sion remained at a similar level. Unexpectedly, the EMT
cells maintained a small spindle-shaped morphology reverse process MET-related gene E-cadherin expression
(Figure 1). was upregulated (Figure 2).
In comparison with DMSO-treated control hiPSCs, four-
3.2. VPA Promoted the EMT-Related Gene Expression. To to eight-day VPA treatment significantly upregulated the
examine whether, or not, VPA could induce hiPSCs to meso- expression of the EMT-related gene N-cadherin and
8 BioMed Research International

vimentin, and the pluripotency-related transcription factor Table 2: Statistical analysis of LVEF at the end of 12 weeks.
Sox2 was downregulated, while the Oct4 expression
remained at a similar level. The EMT reverse process MET- LVEF P value
related E-cadherin gene expression was upregulated at the PBS-injection 36:94 ± 8:31%
P < 0:001
same time. Two-injection 57:9 ± 4:8%
PBS-injection 36:94 ± 8:31%
3.3. Differentiation Induction and Surface Marker Expression P < 0:001
of hiPSC-MSC. Cells at passage 6 were cultured with the Four-injection 64:7 ± 2:4%
chondrogenic differentiation medium for two weeks, and Two-injection 57:9 ± 4:8%
sulphated proteoglycans produced by cell plaques could be P < 0:05
Four-injection 64:7 ± 2:4%
detected using Alcian Blue staining. When cells were cultured
in the adipogenic induction medium, intercellular lipid vacu-
oles could be seen since day 10 by Oil-Red-O staining
(Figure 3). More than 98% of passage 6 hiPSC-MSCs Table 3: Statistical analysis of LVFS at the end of 12 weeks.
expressed typical MSC surface markers (CD29, CD44, LVFS P value
CD73, CD90, and CD105), while the expression of markers
PBS-injection 19:75 ± 3:8%
of hematopoietic cell lineages (CD34, CD45, and HLA-DR) P < 0:001
Two-injection 30:67 ± 3:7%
was below 0.4% (Figure 4).
More than 98% of passage 6 hiPSC-MSCs expressed PBS-injection 19:75 ± 3:8%
P < 0:001
typical MSC surface markers (CD29, CD44, CD73, Four-injection 34:25 ± 5:4%
CD90, and CD105), while the expression of markers of Two-injection 30:67 ± 3:7%
P > 0:05
hematopoietic cell lineages (CD34, CD45, and HLA-DR) Four-injection 34:25 ± 5:4%
was below 0.4%.

3.4. hiPSC-MSC Produced Angiogenesis and Immune Table 4: Statistical analysis of LVEDD at the end of 12 weeks.
Regulatory Factors. Angiogenic and immune regulatory fac- LVEDD(mm) P value
tor secretion level of hiPSC-MSCs was compared with that
PBS-injection 9:36 ± 0:77
of UC-MSCs. UC-MSCs expressed IGF, VEGF, HGF, IDO, P > 0:05
Two-injection 8:67 ± 0:86
PGE2, and IL-10. The expression level of IGF, VEGF, IDO,
PGE2, and IL-10 of hiPSC-MSCs did not significantly differ PBS-injection 9:36 ± 0:77
P > 0:05
from that of UC-MSCs, but the HGF expression level of Four-injection 8:02 ± 0:34
hiPSC-MSCs was significantly lower compared with that of Two-injection 8:67 ± 0:86
P > 0:05
UC-MSCs, P < 0:001 (Figure 5). Four-injection 8:02 ± 0:34
The expression level of VEGF, IGF, IDO, PGE2, and IL-
10 of hiPSC-MSCs was not significantly different from that
of UC-MSCs; however, the HGF expression level of hiPSC- model rats was undertaken, and no tumor formation or evi-
MSCs was significantly lower compared with that of UC- dent pathological changes were identified.
MSCs, ∗∗∗ P < 0:001.
3.6. hiPSC-MSC Administrations Improved the Cardiac
3.5. hiPSC-MSC Dosages and Adverse Effects. In our prelimi- Function. Cardiac function improvement was analyzed by
nary experiment, severe dyspnea was found in some rats monitoring LVEF obtained at week 0 (the day on which the
immediately after intravenous infusions of 2 – 3 × 106 first hiPSC-MSC injection was given) and weeks 2, 4, 6, 8,
hiPSC-MSCs, and these rats died quickly (>30-50%), perhaps 10, and 12. From week 0 to week 12 before sacrificing the rats,
due to thromboemboli in the lungs formed by hiPSC-MSCs, LVEF of the sham group kept unchanged; in the PBS-
as reported by other researchers [16, 17, 33]. As a result, we injection group, LVEF continued to decline, from 48:36 ±
reduced the dose of each injection to 1 × 106 cells, and used 7:30% to 36:94 ± 8:31%; in the two-injection group, LVEF
cells within six passages, to ensure that cells retained their increased from 47:44 ± 7:6% at week 0 to 57:9 ± 4:8% at week
small, spindle-like morphology, thereby facilitating their pas- 12; in the four-injection group, LVEF grew from 46:67 ±
sage through small blood vessels in the lungs. 4:4% at week 0 to 64:7 ± 2:4% at week 12 (Figure 6(a)). Sig-
MSCs are immune-privileged and can be transplanted nificant differences were found when comparing MSC-
into unrelated recipients [3]. In order to test the immune tol- injection groups with the PBS-injection group at the end of
erant and immunosuppressive effects of the induced hiPSC- 12 weeks (PBS-injection group v. two-injection group, P <
MSCs, we used immune competent rats in this study. During 0:001; PBS group v. four-injection group, P < 0:001). There
the four rounds of infusions of 1 × 106 hiPSC-MSCs to model was a significant difference between the two-injection group
rats and the whole 12-week period, symptoms relating to and the four-injection group, P < 0:05 (Figure 6(b) and
acute or chronic immunoreactions, such as dyspnea, convul- Table 2).
sion, oedema, skin rash, langour, or progressive emaciation, LVFS is an important indicator for evaluating the LV sys-
were monitored: no perceptible symptoms were found. At tolic function. From week 0 to week 12, LVFS of the sham
the end of the twelfth week, anatomical examination of the group kept stable; in the PBS-injection group, LVFS declined
BioMed Research International 9

(A) (B) (C)

⁎⁎
20

15

Scar area (%)

(D) (E)
10

PBS-injection
4-injection

2-injection
(a) (b)

Figure 7: hiPSC-MSC administration reduced the scar area. (a) Masson’s Trichrome staining, scar tissue was stained blue, and the viable area
was pink. A. Normal heart. B. Sham infarcted heart. C. PBS-injection-treated AMI heart. D. Two-injection-treated AMI heart. E. Four-
injection-treated AMI heart. (b) MSC treatments reduced the scar area in the cell-injection groups compared with those in the PBS-
injection group, and there was significant difference in the four-injection group, ∗∗ P < 0:01).

from 25:12 ± 2:53% at week 0 to 19:75 ± 3:8% at week 12; in Table 5: Statistical analysis of scar size at the end of 12 weeks.
the two-injection group, LVFS increased from 27:64 ± 2:34%
at week 0 to 30:67 ± 3:7% at week 12; in the four-injection Scar size P value
group, LVFS ascended from 27:97 ± 2:34% at week 0 to PBS-injection 13:74 ± 3:4%
34:25 ± 5:4% at week 12 (Figure 6(c)). Significant differences P > 0:05
Two-injection 10:18 ± 2:16%
were observed upon comparison of the PBS-injection group PBS- injection 13:74 ± 3:4%
with the MSC-injection groups at week 12 (PBS-injection P < 0:01
Four- injection 7:08 ± 3:68%
group v. two-injection group, P < 0:001; PBS-injection group
Two- injection 10:18 ± 2:16%
v. four-injection group, P < 0:001), and there was no signifi- P > 0:05
Four- injection 7:08 ± 3:68%
cant difference between the four-injection group and the
two-injection group (Figure 6(d) and Table 3).
LVEDD was measured continuously after two to four difference in the four-injection group, P < 0:01 (Figure 7(b)
rounds of hiPSC-MSC infusions over 12 weeks. In the sham and Table 5).
group, no significant variation of LVEDD was found; in the
PBS-injection group, LVEDD continued to increase from 3.8. hiPSC-MSC Administrations Promoted Vascularisation
7:8 ± 0:71 mm at week 0 to 9:36 ± 0:77 mm at week 12. In in the Scar Area. Using anti-α-SMA antibody staining, we
the MSC-injection groups, cell treatment prevented LVEDD found that injections of hiPSC-MSC increased the number
increase to different extents from week 0 to week 12 com- of α-SMA+ artery vessels in the scar area of the MSC-
pared with the PBS-injection group (two-injection group: injection groups compared with that of the PBS-injection
from 7:65 ± 0:69 mm to 8:67 ± 0:86 mm; four-injection group (PBS-injection group: 6:12 ± 1:24 v. two-injection
group: from 7:5 ± 0:52 mm to 8:02 ± 0:34 mm) group: 8:625 ± 1:06, P < 0:05; PBS-injection group: 6:12 ±
(Figure 6(e)), despite the insignificant differences among 1:24 v. four-injection group: 13:62 ± 2:19, P < 0:001). There
these groups (Figure 6(f) and Table 4). was a significant difference when comparing two- and four-
injection groups, P < 0:001 (Figure 8 and Table 6).
3.7. hiPSC-MSC Administration Reduced the Scar Size. To 3.9. hiPSC-MSC Administrations Reduced Apoptotic Cell
analyze the potential effect of hiPSC-MSC therapy on the Number in the Border and Infracted Area. Representative
scar size in the AMI model, we stained sections of the scar TUNEL staining images (cells with brown stain) of the
area with Masson’s trichrome. Twelve weeks after AMI, his- four groups are shown, respectively, in Figure 9a. The
tology showed that scar tissue formation in the infarct area results indicated that the cell number of apoptosis in the
was composed mainly of the fibrotic tissue with some inflam- border and infracted area was reduced in the four- and
matory cells, and the scar tissue was stained blue, and the via- two-injection groups (74:53 ± 7:42 and 97:33 ± 8 cells/hpf
ble area was pink (Figure 7(a)): PBS-injection group, ) compared with that of the PBS-injection control group
13:74 ± 3:4%; two-injection group, 10:18 ± 2:16%; and four- (129:33 ± 33:61 cells/hpf), and significant reduction could
injection group, 7:08 ± 3:68%. MSC treatments reduced the be found in the comparison between the four-injection
scar area in the cell-injection groups compared with those group and the PBS-injection group, ∗∗ P < 0:01 (Figure 9b
in the PBS-injection group, and there was a significant and Table 7).
10 BioMed Research International

⁎⁎⁎
20
⁎⁎⁎

Number of artery
15
⁎⁎
10

PBS-injection
4-injection

2-injection
(a) (b)

Figure 8: hiPSC-MSC administration stimulated angiogenesis in the scar area. (a) Detection of blood vessel by anti-α-SMA
immunohistochemistry staining. A α-SMA+ blood vessels in a section of the normal rat (no scar area). B α-SMA+ blood vessels in a
section of the sham operated rat (no scar area). C α-SMA+ blood vessels in a section of the four-injection-treated rat (in scar area). D α-
SMA+ blood vessels in a section of the two-injection-treated rat (in scar area). E α-SMA+ blood vessels in a section the of PBS-injection-
treated rat (in scar area). F Negative staining control, 200×. (b) The number/hpf of α-SMA+ blood vessels in the scar area of the cell-
treated groups was increased significantly compared with that of the PBS-injection group (PBS-injection group v. two-injection group, ∗∗ P
< 0:01; PBS-injection group v. four-injection group, ∗∗∗ P < 0:001). Significant difference could be found when comparing the four-
injection group with the two-injection group, ∗∗∗ P < 0:001.

impotent role in epithelial-to-mesenchymal transition

Table 6: Statistical analysis of angiogenesis at the end of 12 weeks. induction [27, 31], and SB431542 is reported to be feasible in
α-SMA+ artery number/hpf P value MSC derivation from hiPSCs, but it still takes more than
twenty days to generate MSC-like cells [27].
PBS-injection 6:12 ± 1:24
P < 0:05 Through modifying histone acetylation, VPA greatly
Two-injection 8:625 ± 1:06
accelerated the EMT process of hiPSC, and large number of
PBS-injection 6:12 ± 1:24 MSC like cells could be obtained within 15 days in our
P < 0:001
Four-injection 13:62 ± 2:19 research. Compared with the spontaneous commitment
Two-injection 8:625 ± 1:06 and SB431542 induction method, the VPA treatment
P < 0:001
Four-injection 13:62 ± 2:19 method of obtaining MSC is effective and timesaving. The
derived cells expressed MSC-related marker, secreted angio-
genic and immune regulatory factors in levels comparable
4. Discussion to that of UC-MSCs, except for HGF, and could be induced
to experience chondrogenic and adipogenic differentiation,
MSC infusions have been investigated as a treatment means and more importantly did not form a tumor in vivo.
for cardiac disease for more than a decade. Although accu- During the induction process, the expression of EMT-
mulating evidence suggests that the MSC infusion can related genes N-cadherin and vimentin was upregulated to
improve the cardiac function [34], however, information on a very high level, and the expression of pluripotency-related
the long-term dynamic outcomes of multiple hiPSC-MSC genes Sox2 and Oct4 was downregulated or remained
infusions on cardiac disease is unavailable. unchanged, indicating that VPA initiated the process of
The current study is based on the concept that a persis- EMT through upregulating EMT-promoting genes and
tent, excessive inflammatory response contributes to pro- downregulating pluripotency-related gene expression.
gressive myocardial dysfunction post-AMI, and the chronic Although the EMT opposite process MET-related gene E-
inflammatory response cannot be suppressed permanently cadherin expression was upregulated for unspecified reasons,
by a single infusion of MSCs. under the very high level expression of N-cadherin and
Human-induced pluripotent stem cells are an attractive vimentin, the upregulated E-cadherin expression could not
stem cell source for cell therapy. Spontaneous mesoderm com- prevent the initiation of EMT in the VPA-treated cells.
mitment of hiPSCs can be initiated by manually dissociation It has been reported that VPA might activate and stabi-
and followed by subsequent passaging or embryoid body for- lize neurons and astrocytes by stimulating the expression of
mation. It usually takes more than 30 days to generate cells trophic factors, and recent reports indicate that VPA treat-
with increased MSC marker expression and bilineage mesen- ment could enhance multipotency, migration, proliferation,
chymal differentiation potential from hiPSCs [27, 35–37]. and differentiation of MSCs [28, 29]. In our research, we
Small molecules, such as SB431542 and VPA, play an found that low concentration (0.1-0.5 mM) of VPA could
BioMed Research International 11

200 ⁎⁎

Apoptotic cell number

150

100

50

PBS-injection
4-injection

2-injection
(a) (b)

Figure 9: hiPSC-MSC administration reduced the apoptotic cell number in the infarcted and border area. (a) Detection of apoptotic cells by
TUNEL staining. A Apoptotic cell was rarely found in sections of the sham operated rats. B Apoptotic cells in a section of the PBS-injection-
treated rat. C Apoptotic cells in a section of the two-injection-treated rat. D Apoptotic cells in a section of the four-injection treated rat 400×.
(b) The number of apoptotic cells in the infracted and border area of the cell-treated groups was reduced compared with that of the PBS-
injection group, and there was a significant difference between the four-injection group and the PBS-injection group (∗∗ P < 0:01).

Table 7: Statistical analysis of cell apoptosis at the end of 12 weeks. the following two to six weeks, because evidence suggests that
Apoptotic cell number/hpf P value application of MSCs seven days after AMI is preferable to
that given one day after AMI due to differences in the cardiac
PBS-injection 129:33 ± 33:61
P > 0:05 environment after AMI [16]. During the four rounds of infu-
Two-injection 97:33 ± 8
sions of 1 × 106 hiPSC-MSCs to rats, no perceptible adverse
PBS-injection 129:33 ± 33:61 effect and hiPSC-MSC-induced tumor formation were found
P < 0:01
Four-injection 74:53 ± 7:42 over 12 weeks.
Two-injection 97:33 ± 8 LVEF and LVFS are the most commonly used parameters
P > 0:05
Four-injection 74:53 ± 7:42 for evaluating cardiac performance in clinical practice. LV
functional deterioration following AMI remains incomplete
after a few days [39]. LVEDD is a predictor of ventricular
increase MSC viability, whereas concentrations that exceed remodeling [40]. The pathophysiological changes of ventric-
0.5 mM would inhibit cell proliferation and induce cell ular remodeling can be divided into early and late phases
apoptosis in a dosage-dependent manner (data not shown) after AMI onset. The early phase involves expansion of the
indicating its bifunctional property. infarct area, and late remodeling involves time-dependent
Since VPA initiate EMT through modifying histone acety- dilatation, distortion of ventricular shape, and mural hyper-
lation (epigenetic alteration), we did not perform karyotype trophy [41].
analysis on these hiPSC-induced MSCs, and tumor formation We demonstrated that, after AMI induction, without
monitoring only lasted twelve weeks; hence, we do not know intervention, the LVEF, LVFS, and LVEDD of the PBS-
whether the injected cells would form tumor in a longer period. injection control group continued to deteriorate over 12
Tissue origin greatly affects stem cell differentiation and weeks. hiPSC-MSC treatment improved LVEF and LVFS
function of the derived cells. The tissue-specific epigenetic and delayed the increase of LVEDD in the cell-treated
profiles, such as DNA methylation (DNAm) patterns, may groups. LVEDD increases among differently treated groups
not be erased and reestablished completely during repro- were insignificant within a 12-week period, perhaps due to
gramming of somatic cells into hiPSCs. This specific charac- ventricular remodeling being a chronic process.
teristic also remains upon redifferentiation into hiPSC-MSCs The reasons for the poorer improvement of the cardiac
[38]. The low-level secretion of HGF in hiPSC-MSC may be function in the two-injection group may be because, without
related to the incomplete erasing of DNA methylation continuous provision of hiPSC-MSCs to model rats, the ben-
pattern during the reprogramming process. eficial factors secreted by the hiPSC-MSCs were exhausted
Intravenous infusions of hiPSC-MSCs to rats were two to three weeks after the second cell injection; thus, the
started one week after AMI induction and continued for uncurbed inflammation continued for a period of time. With
12 BioMed Research International

a short lifespan, the injected hiPSC-MSCs could not survive of acute myocardial infarction,” The New England Journal of
or multiply for more than three weeks [25, 26]. In addition, Medicine, vol. 362, no. 23, pp. 2155–2165, 2010.
the infused heterogenic hiPSC-MSCs could have been [3] A. Gebler, O. Zabel, and B. Seliger, “The immunomodulatory
cleared gradually by a system of phagocytosis, although it is capacity of mesenchymal stem cells,” Trends in Molecular
known that heterologous MSCs can evade the immune sys- Medicine, vol. 18, no. 2, pp. 128–134, 2012.
tem. This phenomenon emphasises the importance of multi- [4] D. C. Vela, G. V. Silva, J. A. Assad et al., “Histopathological
ple rounds of MSC infusions on sustaining cardiac function study of healing after allogenic mesenchymal stem cell delivery
improvement. in myocardial infarction in dogs,” The Journal of Histochemis-
Van Dijk and Sheu et al. showed that injections of adi- try and Cytochemistry, vol. 57, pp. 167–176, 2008.
pose tissue-derived MSCs could promote angiogenesis and [5] E. C. Perin, M. Tian, F. C. Marini et al., “Imaging long-term
reduce the scar area significantly after AMI [16, 42]. In our fate of intramyocardially implanted mesenchymal stem cells
experiment, the apoptotic cell number of the cell-treated in a porcine myocardial infarction model,” PLoS One, vol. 6,
groups was reduced; the total α-SMA+ artery number in the no. 9, p. e22949, 2011.
scar area in the cell-treated groups was increased, and the size [6] A. R. Williams and J. M. Hare, “Mesenchymal stem cells: biol-
of this area was shrunk significantly compared with that in ogy, pathophysiology, translational findings, and therapeutic
the control group. These results are consistent with that of implications for cardiac disease,” Circulation Research,
vol. 109, pp. 923–940, 2011.
Van Dijk and Sheu et al., indicating that the increased angio-
genesis played an important role in reducing the scar area [7] S. L. Chen, W. W. Fang, F. Ye et al., “Effect on left ventricular
and improving the cardiac function. function of intracoronary transplantation of autologous bone
marrow mesenchymal stem cell in patients with acute myocar-
In conclusion, we demonstrated that MSC-like cells
dial infarction,” The American Journal of Cardiology, vol. 94,
could be effectively induced from hiPSCs by simple VPA no. 1, pp. 92–95, 2004.
treatment, and multiple intravenous infusions of hiPSC-
[8] V. Schächinger, S. Erbs, A. Elsässer et al., “Intracoronary bone
MSCs to AMI rats were an efficacious and safe measure,
marrow-derived progenitor cells in acute myocardial infarc-
whereby to restore reduced cardiac function. tion,” The New England Journal of Medicine, vol. 355, no. 12,
pp. 1210–1221, 2006.
Data Availability [9] G. P. Meyer, K. C. Wollert, J. Lotz et al., “Intracoronary bone
marrow cell transfer after myocardial infarction: eighteen
All the data used to support the findings of this study are months’ follow-up data from the randomized, controlled
included within the article. BOOST (bone marrow transfer to enhance ST-elevation
infarct regeneration) trial,” Circulation, vol. 113, no. 10,
Conflicts of Interest pp. 1287–1294, 2006.
[10] J. M. Hare, J. H. Traverse, T. D. Henry et al., “A randomized,
The authors declare that they have no competing interests double-blind, placebo-controlled, dose-escalation study of
with any individual or institution. intravenous adult human mesenchymal stem cells (prochy-
mal) after acute myocardial infarction,” Journal of the
American College of Cardiology, vol. 54, pp. 2277–2286,
Authors’ Contributions 2009.
Shuyuan Guo and Yusen Zhang contributed equally to this [11] V. Jeevanantham, M. Butler, A. Saad, A. Abdel-Latif, E. K.
work. Zuba-Surma, and B. Dawn, “Adult bone marrow cell therapy
improves survival and induces long-term improvement in car-
diac parameters a systematic review and meta-analysis,” Circu-
Acknowledgments lation, vol. 126, no. 5, pp. 551–568, 2012.
[12] T. Kinnaird, E. Stabile, M. S. Burnett, and S. E. Epstein, “Bone-
We thank Dr. Tiancheng Zhou at Guangzhou Institute of marrow-derived cells for enhancing collateral development:
Biomedicine & Health, Chinese Academy of Sciences for mechanisms, animal data, and initial clinical experiences,” Cir-
kindly providing the human iPSC cell line (PBMC-5). This culation Research, vol. 95, no. 4, pp. 354–363, 2004.
work was supported by the Shenzhen Science and Technol- [13] T. Kinnaird, E. Stabile, M. S. Burnett et al., “Marrow-derived
ogy Innovation Committee [grant number: stromal cells express genes encoding a broad spectrum of
JCYJ20180507183224565 and JCYJ20170816105345191] arteriogenic cytokines and promote in vitro and in vivo arter-
and National Natural Science Foundation of China [grant iogenesis through paracrine mechanisms,” Circulation
number: 81871358]. Research, vol. 94, no. 5, pp. 678–685, 2004.
[14] F. van den Akker, S. C. A. de Jager, and J. P. G. Sluijter, “Mes-
References enchymal stem cell therapy for cardiac inflammation: immu-
nomodulatory properties and the influence of toll-like
[1] C. Toma, M. F. Pittenger, K. S. Cahill, B. J. Byrne, and P. D. receptors,” Mediators of Inflammation, vol. 2013, Article ID
Kessler, “Human mesenchymal stem cells differentiate to a 181020, 13 pages, 2013.
cardiomyocyte phenotype in the adult murine heart,” Circula- [15] D. Kyurkchiev, I. Bochev, E. Ivanova-Todorova et al., “Secre-
tion, vol. 105, no. 1, pp. 93–98, 2002. tion of immunoregulatory cytokines by mesenchymal stem
[2] R. W. Yeh, S. Sidney, M. Chandra, M. Sorel, J. V. Selby, and cells,” World Journal of Stem Cells, vol. 6, no. 5, pp. 552–570,
A. S. Go, “Population trends in the incidence and outcomes 2014.
BioMed Research International 13

[16] A. van Dijk, B. A. Naaijkens, W. J. F. M. Jurgens et al., “Reduc- blood mesenchymal stromal cells treated with histone deacety-
tion of infarct size by intravenous injection of uncultured adi- lase inhibitor valproic acid,” Stem Cells International,
pose derived stromal cells in a rat model is dependent on the vol. 2014, Article ID 610495, 14 pages, 2014.
time point of application,” Stem Cell Research, vol. 7, no. 3, [30] J. Xu, S. Lamouille, and R. Derynck, “TGF-β-induced epithe-
pp. 219–229, 2011. lial to mesenchymal transition,” Cell Research, vol. 19, no. 2,
[17] T. Mäkelä, R. Takalo, O. Arvola et al., “Safety and biodistribu- pp. 156–172, 2009.
tion study of bone marrow-derived mesenchymal stromal cells [31] Y. M. Zhang, Y. S. Zhang, M. H. Li et al., “Combination of
and mononuclear cells and the impact of the administration SB431542, CHIR99021 and PD0325901 has a synergic effect
route in an intact porcine model,” Cytotherapy, vol. 17, no. 4, on abrogating valproic acid-induced epithelial-mesenchymal
pp. 392–402, 2015. transition and stemness in HeLa, 5637 and SCC-15 cells,”
[18] A. Zanetti, M. Grata, E. B. Etling, R. Panday, F. S. Villanueva, Oncology Reports, vol. 41, no. 6, pp. 3545–3554, 2019.
and C. Toma, “Suspension-expansion of bone marrow results [32] G. H. Cui, Z. Qi, X. Guo, J. Qin, Y. Gui, and Z. Cai, “Rat bone
in small mesenchymal stem cells exhibiting increased trans- marrow derived mesenchymal progenitor cells support mouse
pulmonary passage following intravenous administration,” ES cell growth and germ-like cell differentiation,” Cell Biology
Tissue Engineering. Part C, Methods, vol. 21, no. 7, pp. 683– International, vol. 33, no. 3, pp. 434–441, 2009.
692, 2015.
[33] D. Furlani, M. Ugurlucan, L. L. Ong et al., “Is the intravascular
[19] J. Galipeau and L. Sensébé, “Mesenchymal stromal cells: clini-
administration of mesenchymal stem cells safe?: Mesenchymal
cal challenges and therapeutic opportunities,” Cell Stem Cell,
stem cells and intravital microscopy,” Microvascular Research,
vol. 22, no. 6, pp. 824–833, 2018.
vol. 77, no. 3, pp. 370–376, 2009.
[20] X. Q. Tian, Y. J. Yang, Q. Li et al., “Combined therapy with
atorvastatin and atorvastatin-pretreated mesenchymal stem [34] B. Vrtovec and R. Bolli, “Potential strategies for clinical trans-
cells enhances cardiac performance after acute myocardial lation of repeated cell therapy,” Circulation Research, vol. 124,
infarction by activating SDF-1/CXCR4 axis,” American Jour- no. 5, pp. 690–692, 2019.
nal of Translational Research, vol. 11, no. 7, pp. 4214–4231, [35] C. Xu, J. Jiang, V. Sottile, J. McWhir, J. Lebkowski, and M. K.
2019. Carpenter, “Immortalized fibroblast-like cells derived from
[21] L. Woudstra, P. A. J. Krijnen, S. J. P. Bogaards et al., “Develop- human embryonic stem cells support undifferentiated cell
ment of a new therapeutic technique to direct stem cells to the growth,” Stem Cells, vol. 22, no. 6, pp. 972–980, 2004.
infarcted heart using targeted microbubbles: StemBells,” Stem [36] N. S. Hwang, S. Varghese, H. J. Lee et al., “In vivo commitment
Cell Research, vol. 17, no. 1, pp. 6–15, 2016. and functional tissue regeneration using human embryonic
[22] S. Fickert, U. Schröter-Bobsin, A. F. Groß et al., “Human mes- stem cell-derived mesenchymal cells,” Proceedings of the
enchymal stem cell proliferation and osteogenic differentiation National Academy of Sciences, vol. 105, no. 52, pp. 20641–
during long-term ex vivo cultivation is not age dependent,” 20646, 2008.
Journal of Bone and Mineral Metabolism, vol. 29, no. 2, [37] S. E. Brown, W. Tong, and P. H. Krebsbach, “The derivation of
pp. 224–235, 2011. mesenchymal stem cells from human embryonic stem cells,”
[23] A. Stolzing, E. Jones, D. McGonagle, and A. Scutt, “Age-related Cells, Tissues, Organs, vol. 189, no. 1-4, pp. 256–260, 2008.
changes in human bone marrow-derived mesenchymal stem [38] J. Frobel, H. Hemeda, M. Lenz et al., “Epigenetic rejuvenation
cells: consequences for cell therapies,” Mechanisms of Ageing of mesenchymal stromal cells derived from induced pluripo-
and Development, vol. 129, no. 3, pp. 163–173, 2008. tent stem cells,” Stem Cell Reports, vol. 3, no. 3, pp. 414–422,
[24] S. Zhou, J. S. Greenberger, M. W. Epperly et al., “Age-related 2014.
intrinsic changes in human bonemarrow-derived mesenchy- [39] M. A. Konstam, J. E. Udelson, I. S. Anand, and J. N. Cohn,
mal stem cells and their differentiation to osteoblasts,” Aging “Ventricular remodeling in heart failure: a credible surrogate
Cell, vol. 7, no. 3, pp. 335–343, 2008. endpoint,” Journal of Cardiac Failure, vol. 9, no. 5, pp. 350–
[25] S. Kern, H. Eichler, J. Stoeve, H. Klüter, and K. Bieback, “Com- 353, 2003.
parative analysis of mesenchymal stem cells from bone mar- [40] M. A. Konstam, D. G. Kramer, A. R. Patel, M. S. Maron, and
row, umbilical cord blood, or adipose tissue,” Stem Cells, J. E. Udelson, “Left ventricular remodeling in heart failure:
vol. 24, no. 5, pp. 1294–1301, 2006. current concepts in clinical significance and assessment,”
[26] K. Bieback, S. Kern, A. Kocaomer, K. Ferlik, and P. Bugert, JACC: Cardiovascular Imaging, vol. 4, no. 1, pp. 98–108, 2011.
“Comparing mesenchymal stromal cells from different human [41] Y. Inaba, B. P. Davidson, S. Kim et al., “Echocardiographic
tissues: bone marrow, adipose tissue and umbilical cord evaluation of the effects of stem cell therapy on perfusion
blood,” Bio-medical Materials and Engineering, vol. 18, and function in ischemic cardiomyopathy,” Journal of the
pp. 71–76, 2008. American Society of Echocardiography, vol. 27, no. 2,
[27] Y. S. Chen, R. A. Pelekanos, R. L. Ellis, R. Horne, E. J. Wolve- pp. 192–199, 2014.
tang, and N. M. Fisk, “Small molecule mesengenic induction of [42] J. J. Sheu, M. S. Lee, C. G. Wallace et al., “Therapeutic effects of
human induced pluripotent stem cells to generate mesenchy- adipose derived fresh stromal vascular fraction-containing
mal stem/stromal cells,” Stem Cells Translational Medicine, stem cells versus cultured adipose derived mesenchymal stem
vol. 1, no. 2, pp. 83–95, 2012. cells on rescuing heart function in rat after acute myocardial
[28] S. Y. Um, H. Lee, Q. Zhang, H. Y. Kim, J. H. Lee, and B. M. Seo, infarction,” American Journal of Translational Research,
“Valproic acid modulates the multipotency in periodontal liga- vol. 11, no. 1, pp. 67–86, 2019.
ment stem cells via p53-mediated cell cycle,” Tissue Engineering
and Regenerative Medicine, vol. 14, no. 2, pp. 153–162, 2017.
[29] L. A. Marquez-Curtis, Y. Qiu, A. Xu, and A. Janowska-Wiec-
zorek, “Migration, proliferation, and differentiation of cord
 Copyright © 2020 Shuyuan Guo et al. This is an open access article
distributed under the Creative Commons Attribution License (the “License”),
which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited. Notwithstanding the ProQuest
Terms and Conditions, you may use this content in accordance with the terms
of the License. https://creativecommons.org/licenses/by/4.0/