Elham Hoveizi1,2, S Hima Tavakol3. 1. Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Email: e.hoveizi@scu.ac.ir. 2. Stem Cells and Transgenic Technology Research Center (STTRC), Shahid Chamran University of Ahvaz, Ahvaz, Iran. 3. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
Treatment of chronic endoderm-derived organ failures,
such as hepatic cirrhosis and diabetes, is rather challenging
since no complete cure is known (1). Today, organ
transplantation is the major treatment for these disorders.
However, it is accompanied with many difficulties,
including shortage of donors, transplant rejections,
surgical complications, and high costs. Today, cell therapy
has been considered as an alternative approach to remove
some of these barriers (2).Mesenchymal stem cells (MSCs) are multipotent and
differentiate into various cells and tissues, as these cells
have been found in most adult tissues (2, 3). Recently,
the application of human wharton’s jelly MSCs
(hWJMSCs) in therapy has come to attention. Because
of high potency, multipotent properties amongst adult
stem cells, fast and relatively inexpensive extraction,
and their non-tumorigenic state, hWJMSCs have been
suggested as a promising candidate for cell therapy
(4). Suitably for our studies, it has been shown that
hWJMSCs may be induced to differentiate into a variety
of cell types including definitive endoderm (DE), beta
cells, and hepatocytes (5, 6).The first and basic developmental step in formation
of endodermal organs is the induction of DE (7).
Notably, the pancreas, lungs, liver, and other organs of
the gastrointestinal tract are derived from DE during
embryogenesis (8, 9). Multiple signaling pathways such as
Nodal, Activin A, and Wnt3a could cause differentiation
of DE through signaling intermediates. In addition,
the Wnt/β-catenin signaling has a critical role in cell
morphology, proliferation, motility, axis determination,
differentiation, and organ development. SKL2001, as an
agonist for Wnt/β-catenin signaling, has been shown to
inhibit the phosphorylation of β-catenin (by an alternative
mechanism and independent of inhibition of GSK-3β activity), thus increasing the levels of intracellular
β-catenin (10).According to previous studies, zinc is considered as an
abundant trace metal and a catalyzer for various enzymes in the body (11, 12). Besides, zinc protects insulin
from degradation and stimulates insulin biosynthesis,
secretion, and its storage. Different zinc transporters,
such as zinc transporter-8, exist in pancreatic β-cells
and affect insulin secretion. Also, zinc improves
insulin signaling by several mechanisms, such as
increasing insulin receptor phosphorylation, inhibition
of glycogen synthase kinase-3 (GK), and increasing
PI3K activity (11). Indeed, based on recent reports, zinc
oxide nanoparticles (nZnO) reduce blood glucose and
significantly increase blood insulin in diabetic animal
models, when compared to ZnO (12).On the other hand, not only intracellular signaling
pathways, but also cell interactions with the extracellular
matrix (ECM) are taken as critical parameters in behavior,
adhesion, morphology, migration, proliferation, and
differentiation of the cells (13). In tissue engineering,
a suitable scaffold can provide this platform. Also, the
electrospinning technology can be applied to fabricate
three-dimensional (3D) synthesized scaffolds. The
electrospun scaffolds with diameters of tens to hundreds
of nanometers are designed to mimic the ECM in cell and
tissue culture (10, 14).Thus in this study, we used SKL2001 as a Wnt/β-catenin pathway agonist alone or in
combination with nZnO to induce differentiation of hWJMSCs into SOX17-
expressing cells (as DE like-cells) on PLA/Cs three-dimensional scaffolds.
Materials and Methods
All animal procedures and experimental tests were
approved by the Animal Ethics Committee of Shahid
Chamran University of Ahvaz (93042515).
Preparation of scaffold by electrospinning
In this experimental study, the electrospinning
technique was employed to fabricate polylactic acid/
chitosan (PLA/Cs) scaffolds (Electronic, FNM, Iran). To
obtain 2.5 (w/v) solutions, PLA and chitosan (Cs) were
added in hexafluoroisopropanol (HFIP) and acetic acid,
respectively. These solutions were blended in the ratio of
7:3 (PLA:Cs) to make a scaffold. The solution was shaken
for 12 hours and inserted into a 10-ml plastic syringe, and
connected to a high voltage (14-18 kV) at 25°C Aluminum
foil was used to collect spray drift (at a distance of 10
centimeters). The electrospun fibrous membranes were
dried in a vacuum oven for two days and separated from
aluminum foil at the time of use (15, 16).
Cell seeding on PLA/Cs nanocomposite scaffold
The scaffold was cut into discs with a diameter of 1.6 centimeters and placed in 24-well
plates. After that, the scaffolds were sterilized by ultraviolet irradiation for 2 hours
and floated in Dulbecco’s modified eagle medium (DMEM, Gibco, USA) medium supplemented
with 1 µg/ ml amphotericin B and 3% pen/strep overnight at 37°C. Then, hWJMSCs were
cultured at a density of 6×104 cell/scaffold and incubated at 37°C and 5%
CO2 .
Scanning electron microscopy
The morphology of the prepared PLA/Cs scaffolds was
studied with scanning electron microscopy (SEM). The
average diameter of the mats was measured by analyzing
SEM images using Image J software (National Institutes
of Health, USA). The diameter distribution was measured
by examining at least 100 samples. To observe hWJMSCs
cultured on PLA/Cs nanocomposite scaffolds, the
samples were fixed with 2.5% glutaraldehyde for 2 hours.
Then they were washed in phosphate buffer saline (PBS,
Sigma, USA) and dehydrated in ethanol series (30, 50,
70, 80, 90 and 100%) at 37ºC for 15 minutes per solution.
Then the scaffolds were sputter-coated with gold and
studied by a SEM (model Philips XL-30, Netherland),
operated at 15 kV.
Isolation and identification of hWJMSCs
Human WJMSCs were obtained as described previously (17, 18). In summary, human umbilical
cord was obtained after delivery from term natural births. The cord blood was removed
immediately, then it was cut into 1-centimeter pieces and washed. The pieces were soaked
in PBS supplemented with 3% (v/v) pen/strep for 24 hours. The stem cells were isolated by
explant cultures as each piece was cut carefully with a scalpel, then the vessels were
removed and wharton jelly was collected. Then the jelly was sliced into 2-millimeter
pieces, cultured in tissue culture flasks and maintained for 14 days to allow for cell
migration and expansion. DMEM low glucose medium supplemented with 10% fetal bovine serum
(FBS, Gibco, USA) and 1% penicillin/ streptomycin was used and the medium was changed
every 3 days as the cells were passaged by 0.25% trypsin. Also, hWJMSCs were characterized
by flow cytometry for cell surface markers. Briefly, hWJMSCs were incubated with specific
antibodies including CD146 (endometrial stem cell markers, 1:200, Santa Cruz, USA), CD90
(1:200, Santa Cruz, USA), CD105 (1:100, Santa Cruz, USA), CD34 (hematopoietic marker,
1:100, Santa Cruz, USA), and CD31 (endothelial marker, 1:100, Santa Cruz, USA) for 60
minutes at room temperature and analyzed by flow cytometry (Becton Dickinson, USA) after
washing. Also, for differentiation of hWJMSCs into adipocytes and osteocytes, the cells
were seeded at a concentration of 5×104 cells/well in a 24-well plate. When the
cells reached 80% confluency, the medium was replaced with adipogenic- or
osteogenic-inducing media as described previously (17). The differentiated cells were
stained with Oil Red or Alizarin Red to detect adipocytes or osteocytes, respectively.
Assessment of cell viability
The viability of the hWJMSCs seeded on the PLA/ CS scaffolds was assessed by the MTT
(3-(4, 5- Dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide) reduction assay.
6×104 cells were cultured on each scaffold and incubated for 24 hours at 37°C
in an incubator with 5% Co2 . Afterward, 300 µl of 0.5 mg/ml MTT solution was
added to each well, and the plates were incubated for 3-4 hours at 37°C then the medium
was removed, and DMSO was added to dissolve the formazan crystals. The samples were shaken
by a mechanical shaker, next the absorbance was read at the wavelength of 490 nanometers
in a microplate reader (Fax 2100, USA).
Acridine orange staining
For acridine orange/ethidium bromide double staining
(Sigma, USA), 1 mg/ml ethidium bromide dye and 1 mg/
ml acridine orange dye were prepared and mixed at a 1:1
ratio. After that, the cells were seeded on scaffolds (after
48 hours), were stained for 3 minutes and then examined
under a fluorescent microscope (Olympus, Japan).
Human WJMSCs culture and differentiation into
definitive endoderm cells
The cultured cells on PLA/Cs scaffolds were induced to
differentiate using two different protocols. The first: the
hWJMSCs were cultured on PLA/Cs scaffold and treated
with 20 µM SKL2001 (Sigma, USA) and 0.2% FBS for
6 days. The second: the cells were cultured on PLA/Cs
scaffold and treated with 20 µM SKL2001 in combination
with 50 µg/ml nZnO (Loletics Germany, ≥70 nm avg.) for
6 days. As a control, hWJMSCs were cultured on the PLA/
Cs scaffolds in the absence of differentiation factors for 6
days. DMEM medium was used during the differentiation
phase. This medium was supplemented with FBS at 0.2%
and 10% concentrations in the experimental and control
groups, respectively. The medium in cell culture plates
was replaced with fresh medium every two days.
RNA extraction and reverse transcriptase-polymerase
chain reaction
The mRNA expression was examined by quantitative reverse transcriptase-polymerase chain
reaction (qRT-PCR). On day 6 days of the culture, the cells were lysed by QIAzol lysis
reagent (Qiagen, Hilden, Germany) and the total RNA was extracted according to the
manufacturer’s instructions. The extracted RNA (3 μg) was reverse transcribed by the
TaqMan Reverse Transcription Kit (Applied Biosystems, CA, USA). QRT-PCR reactions were
performed in 6-well plates in a StepOne™ Real-Time PCR machine (Corbett, Australia) using
primers that are shown in Table 1. QRT-PCR was performed by SYBR green Supermix (Ampliqon,
Denmark) and the applied protocol was: initial denaturation (95°C for 30 seconds),
amplification (95°C for 5 seconds and 60°C for 33 seconds), and melting (95°C for 15
seconds, 60°C for 60 seconds, and 95°C for 1 second). The target genes’ threshold cycle
(Ct) was obtained from the StepOne software, and the values were normalized by
GAPDH.
Immunofluorescence staining
The samples were fixed with paraformaldehyde (4%, Sigma, USA) for 40 minutes and
permeabilized with 0.1% Triton X-100 in PBS at room temperature. Then the samples were
blocked for 1 hour with 5% bovine serum albumin (BSA, Sigma, USA) at room temperature and
stained with primary antibodies against human FOXA2 (1: 500, Polyclonal
rabbit IgG, Millipore, Germany, AB4125) and human SOX17 (1: 20,
Polyclonal Goat IgG, R&D, USA, AF1924) overnight at 4°C. After that, the samples were
stained with secondary antibodies [Alexa fluor 594 donkey anti-rabbit (1:200, Gibco, USA,
A-21207) or Alexa fluor 488 donkey anti-goat (1:200, Gibco, USA, A-11058)] for 60 minutes
at room temperature. The cell nuclei were stained with DAPI (1 μg/ml, Sigma, USA, D8417)
for 5 minutes and the samples were imaged under a fluorescent microscope (Olympus, Japan).
Statistical analysis
The data are presented as means ± standard deviation
(SD) of three replications. Statistical analyses were
carried out by a one-way ANOVA method followed by
unpaired Student’s t test and P≤0.05 was designated as
significant difference. We used SPSS software version 16.Sequences of the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) primers
Results
Characterization of hWJMS cells
Human WJMSCs readily adhered to the bottom of the
flask and were extractable. After 2 weeks, the cells were
fully attached to the bottom of the flasks and had grown to
80% confluency. At this point they were passaged one last
time and reached to the third passage, after which they were
used for treatment. As shown in Figure 1, under the inverted
microscope, the cells appeared to be normal with elongated
and spindle-like shapes (Fig .1A). To confirm multipotency
of the isolated hWJMSCs, they were treated with osteogenic
and adipogenic inductive media, and their differentiation
was confirmed by Alizarin Red, and Oil Red O staining,
respectively (Fig .1B, C). In addition, when evaluating their
cell surface markers by flow cytometry, our results indicated
that the cells were expressing CD105, CD90, and CD146
markers, but not CD31 and CD34 (Fig .1D).
Fig.1
Characterization of human Wharton’s jelly mesenchymal stem cells (hWJMSCs). A.
Typical observation of hWJMSCs under phase-contrast microscopy. B.
Differentiation of hWJMSCs into osteoblasts as shown by Alizarin Red staining.
C. Differentiation of hWJMSCs into adipocytes as shown by Oil Red O
staining. D. Flow cytometric analysis of the isolated hWJMSCs for MSC
(CD146, CD105, and CD90), and hematopoietic (CD34) and endothelial (CD31) markers
(scale bar: 100 µm).
Characterization of human Wharton’s jelly mesenchymal stem cells (hWJMSCs). A.
Typical observation of hWJMSCs under phase-contrast microscopy. B.
Differentiation of hWJMSCs into osteoblasts as shown by Alizarin Red staining.
C. Differentiation of hWJMSCs into adipocytes as shown by Oil Red O
staining. D. Flow cytometric analysis of the isolated hWJMSCs for MSC
(CD146, CD105, and CD90), and hematopoietic (CD34) and endothelial (CD31) markers
(scale bar: 100 µm).
Morphology of the electrospun PLA/Cs nanocomposite
scaffold
We produced the PLA/Cs nanocomposite scaffolds
by a solution blending method. As shown in Figure 1,
the nanocomposite was a homogeneous scaffold with
high porosity and average diameter of 70 micrometers.
Also, the average thickness of this scaffold was about
460 micrometers. Indeed, the SEM images indicated
random fibers without any beads with an improved porous network. Also, the study of culturing the adherent cells
on the scaffold revealed that numerous hWJMSCs were
attached and scattered on PLA/Cs scaffold after 72 hours
following the seeding (Fig .2). These results proved
that the PLA/Cs scaffold effectively supported cellular
adhesion and growth of the hWJMSCs.
Fig.2
Scanning electron micrographs show the morphology of the plated human Wharton’s jelly mesenchymal
stem cells (hWJMSCs) on polylactic acid/chitosan (PLA/Cs) scaffold on day 3 after
seeding. A. The fibers of PLA/Cs scaffold were randomly entangled to form
a strong, flexible, and porous 3D matrix. B. The fibers of PLA/Cs
scaffold with higher magnification, C. The plated hWJMSCs on PLA/Cs
scaffold, D. The plated hWJMSCs on PLA/Cs scaffold with higher
magnification. Yellow arrows show the plated hWJMSCs on the scaffold.
Scanning electron micrographs show the morphology of the plated human Wharton’s jelly mesenchymal
stem cells (hWJMSCs) on polylactic acid/chitosan (PLA/Cs) scaffold on day 3 after
seeding. A. The fibers of PLA/Cs scaffold were randomly entangled to form
a strong, flexible, and porous 3D matrix. B. The fibers of PLA/Cs
scaffold with higher magnification, C. The plated hWJMSCs on PLA/Cs
scaffold, D. The plated hWJMSCs on PLA/Cs scaffold with higher
magnification. Yellow arrows show the plated hWJMSCs on the scaffold.
Measurement of cell viability
In this study, we evaluated cell viability of hWJMSCs
on PLA/Cs scaffolds using the MTT assay for 6 days.
Three days after plating the cells, no significant
difference was observed between the viability of
cells on PLA/Cs scaffold and the monolayer culture
(P<0.05). However, on days 4 and 6 of the culture, the
viability of the cells on PLA/Cs scaffolds significantly
increased (P<0.05) compared to the monolayer
culture. Therefore, it suggests that there is a time-dependent increase in stability and viability of the
hWJMSCs on the nanocomposite scaffold compared
to the monolayer culture (Fig .3A).
Fig.3
MTT assay and morphological study of hWJMSCs by an inverted microscope. A. Formosan
absorbance has been expressed as a measure of cell viability from the hWJMSCs cultured
on nanocomposite scaffold for 6 days. B. Passage 1 hWJMSCs after 3 days
in culture, C. Passage 3 hWJMSCs after 7 days, D. The cell
viability assay using acridine orange/ ethidium bromide staining of hWJMSCs was
performed on the cells cultured on PLA/Cs scaffold by fluorescent microscopy after 2
days (B, C, and D represent ×200, ×200, and ×100 magnification, respectively, scale
bar: 100 µm). hWJMSCs; Human Wharton’s jelly mesenchymal stem cells, PLA/Cs;
Polylactic acid/chitosan, and ***; P<0.05 and values are mean (n=3).
Acridine orange is a double staining, such that live
cells turn green, while apoptotic cells are orange, and
if cells are in late stages of apoptosis, the nuclei get
fragmented, compacted, and red. Our result suggested
that the cultured cells on the PLA/Cs scaffold were
normal, clear, green and without shrinkage, proving
their viability (Fig .3).MTT assay and morphological study of hWJMSCs by an inverted microscope. A. Formosan
absorbance has been expressed as a measure of cell viability from the hWJMSCs cultured
on nanocomposite scaffold for 6 days. B. Passage 1 hWJMSCs after 3 days
in culture, C. Passage 3 hWJMSCs after 7 days, D. The cell
viability assay using acridine orange/ ethidium bromide staining of hWJMSCs was
performed on the cells cultured on PLA/Cs scaffold by fluorescent microscopy after 2
days (B, C, and D represent ×200, ×200, and ×100 magnification, respectively, scale
bar: 100 µm). hWJMSCs; Human Wharton’s jelly mesenchymal stem cells, PLA/Cs;
Polylactic acid/chitosan, and ***; P<0.05 and values are mean (n=3).
Differentiation of hWJMSCs into definitive endoderm
cells
To distiguish DE cells from hWJMSCs, two protocols (SKL2001 or SKL2001/ nZnO) were used
and their efficiencies were compared. As shown in Figure 4, in response to SKL2001
induction, the expression of the hallmark genes of DE i.e. FOXA2, SOX17,
and GSC were increased about 130, 159, and 90-fold, respectively. Also,
the induction of hWJMSCs with SKL2001/nZnO resulted in a significant increase in the
expression levels of FOXA2, SOX17, and gsc genes to 141,
204, and 92 folds, respectively. The expression levels of FOXA2 and
SOX17 were also significantly higher (P<0.05) than their
expressions in the cells induced by SKL2001 alone, but no obvious difference in
GSC expression was found between these two groups (Fig .4). In this
research, mRNAs expression of the DE marker genes in both experimental groups was
significantly more than that in the control group. Based on these data, treatment with
SKL2001 or especially with SKL2001/nZnO could obviously induce the differentiation of
hWJMSCs into DE cells.
Fig.4
Quantitative expression analysis of DE cells derived from hWJMSCs cultured on PLA/Cs scaffold
after 6 days. The results are collected from 3 independent experiments with 2 internal
replicates per experiment. Differences observed were statistically significant when
P≤0.05. Comparison of the gene expression levels of DE markers (FOXA2,
SOX17, and GSC) in two experimental groups. hWJMSCs; Human
Wharton’s jelly mesenchymal stem cells, PLA/Cs; Polylactic acid/chitosan, DE;
Definitive endoderm, ***; P<0.001, and **; P<0.01 untreated cells were
considered as a control group.
Immunocytochemical technique was performed
for further evaluation of the more effective protocol
i.e. SKL2001/nZnO. This analysis of the hWJMSCs-derived DE sample suggested that the main population
of DE cells expressed SOX17 and FOXA2 proteins
within the nuclei. Therefore, treatment with SKL2001/nZnO could successfully induce differentiation into
DE cells (Fig .5).
Fig.5
Immunocytochemistry performed for analyzing SOX17 and FOXA2
as endoderm-specific proteins by differentiated hWJMSCs on the scaffold
after 6 days of culture. The staining of nuclei was performed by DAPI (×400
magnification, scale bar: 100 µm).
Quantitative expression analysis of DE cells derived from hWJMSCs cultured on PLA/Cs scaffold
after 6 days. The results are collected from 3 independent experiments with 2 internal
replicates per experiment. Differences observed were statistically significant when
P≤0.05. Comparison of the gene expression levels of DE markers (FOXA2,
SOX17, and GSC) in two experimental groups. hWJMSCs; Human
Wharton’s jelly mesenchymal stem cells, PLA/Cs; Polylactic acid/chitosan, DE;
Definitive endoderm, ***; P<0.001, and **; P<0.01 untreated cells were
considered as a control group.Immunocytochemistry performed for analyzing SOX17 and FOXA2
as endoderm-specific proteins by differentiated hWJMSCs on the scaffold
after 6 days of culture. The staining of nuclei was performed by DAPI (×400
magnification, scale bar: 100 µm).
Discussion
In the present study, we have assessed the potentials of differentiation of hWJMSCs into DE
cells using SKL2001 as a small molecule and nZnO as a nanoparticle on an electrospun
nanocomposite scaffold. The analysis of qRT-PCR demonstrated that the expression of
endodermal marker genes such as FOXA2, SOX17, and GSC in
experimental groups were significantly more than the control group. Using
immunocytochemistry, we also evaluated the expression levels of the protein products of
these genes.We can define tissue engineering as a multidisciplinary approach that provides a promising
strategy for regenerative medicine (19). Various research projects have demonstrated the
advantages of tissue engineering as a suitable therapeutic strategy for induction of cell
differentiation (20, 21). Interestingly, these studies have proven that the use of tissue
engineering improves cell proliferation, survival, and cell-cell interactions comparing to
monolayer cultures (22-24). Today, the administration of synthetic polymers to prepare
scaffolds has significantly increased, which is due to their suitable mechanical properties,
cost-effectiveness, and convenient fabrication processes. Furthermore, polymer blending
methods have been used to improve the hydrophilic properties of synthetic polymers and the
adhesion of cells to scaffolds (25-28). In our study, PLA/Cs, as a blended scaffold, was a
biocompatible and suitable scaffold in promoting cell viability and attachment. Also, the
infiltration and extension of the cells into the PLA/ Cs scaffold was confirmed by SEM
studies. Furthermore, according to our results, the DE differentiation with high efficiency
in the expression of SOX17 was due to the treatment of hWJMSCs seeded on
PLA/Cs with SKL2001 and nZnO.It has been proved that the formation of DE can be
considered as the first and most critical stage in the
generation of stem cell-derived hepatocytes, beta cells,
and other endoderm derived organs (29-31). D’Amour et
al. (29) in 2006 presented a protocol for efficient induction
of DE cells. Because of the high efficiency of this protocol,
generally it has been used by many research groups (32,
33). Based on the recent studies, to induce DE cells from
different stem cells, several factors including Nodal,
Wnt3a, Activin A, and some of the small molecules have
been used (31). For example, Borowiak et al. (34) showed
that inducer of DE1 as a small molecule could increase DE
differentiation, comparable to those induced by Activin A.
In the present study, our results indicated that SKL2001/
nZnO combination offers a practical method for DE
differentiation from hWJMSCs in a 3D culture.Previous studies have reported that zinc is effective
in glucose metabolism, promoting hepatic glycogenesis
by acting on insulin pathways and improving glucose
utilization. Zinc plays an essential role in biosynthesis,
secretion, and storage of insulin because it protects insulin structure. Moreover, beta cells contain several zinc
transporters that stimulate insulin secretion (35). Also,
nZnO has antidiabetic effects and it can decrease blood
glucose, inhibit glucokinase (GK) activity, increase insulin
level, and stimulate the expression of glucose transporter
2 in diabetic rats (12). Besides, in some studies, it has been
reported that zinc ions act as signalling molecules (36,
37). Thus, we can divide the intracellular zinc functions
into two categories: i. Protein binding zinc, contributing
to enzyme’s activity and structure protection, and ii.
Labile zinc, with is non-binding to proteins and acts as a
signal transferring molecule (38). Based on the existing
evidence, nZnO may be conducive to the differentiation of
DE through signaling pathways, especially by inhibiting
GK activity. In this study we showed that the small
molecule SKL2001 has synergistic effects with nZnO in
induction of DE cell formation. We conclude that in this
induction pathway, nZnO has synergies with SKL2001
via activation of β-catenin signaling, since nZnO can
activate Wnt/β-catenin indirectly by inhibiting GK.The Wnt/β-catenin has important functions in
differentiation processes of various cells such as MSCs.
Gwak et al. (39) in 2012 introduced SKL2001 as a novel
agonist of the Wnt/β-catenin pathway after screening 270
000 synthetic chemical compounds. They identified the
molecular mechanism of SKL2001, which leads to release
of β-catenin, by opening up the Axin/β-catenin bond.Maschio et al. declared that Wnt/β-catenin agonist plays
a crucial function in cell differentiation and proliferation
during embryogenesis. They specifically demonstrated
that there is a relationship between the Wnt/β-catenin
pathway and type 2 diabetes. Also, they found that
the unregulated Wnt/β-catenin pathway leads to the
disruption of beta cells in the early phase of diabetes (40).
Conclusion
Our findings indicated that the PLA/Cs nanocomposite
scaffolds provide a protective and suitable environment for
hWJMSCs’ growth and viability. Here we showed for the
first time that the small molecule SKL2001 as a Wnt/β-catenin pathway agonist could induce differentiation of
hWJMSCs into DE cells. Also, our results showed that
the treatment of hWJMSCs with SKL2001 combined
with nZnO had a synergistic effect on DE cell induction.
It has been suggested that we can provide an efficient
method with the functional differentiation of DE cells via
combining a suitable scaffold with essential supplements
and a reliable cellular source. These results be used for
further differentiation into pancreatic and hepatocytes cells.
Table 1
Sequences of the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) primers
Authors: Kevin A D'Amour; Alan D Agulnick; Susan Eliazer; Olivia G Kelly; Evert Kroon; Emmanuel E Baetge Journal: Nat Biotechnol Date: 2005-10-28 Impact factor: 54.908
Authors: Qiping Lu; Hariprakash Haragopal; Kira G Slepchenko; Christian Stork; Yang V Li Journal: Int J Physiol Pathophysiol Pharmacol Date: 2016-04-25
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