Improved differentiation of umbilical cord blood-derivedmesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Anh Nguyen-Tu Bui, Loan Thi-Tung Dang, Khue Gia Ngu
Trang 1Improved differentiation of umbilical cord blood-derived
mesenchymal stem cells into insulin-producing cells
by PDX-1 mRNA transfection
Anh Nguyen-Tu Bui, Loan Thi-Tung Dang, Khue Gia Nguyen, Ngoc Kim Phan
Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Viet Nam
a r t i c l e i n f o
Article history:
Received 29 April 2014
Received in revised form
4 August 2014
Accepted 18 August 2014
Keywords:
Mesenchymal stem cells
UCB-MSCs
Insulin producing cells
PDX-1
mRNA transfection
a b s t r a c t
Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group) This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group) We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.372.5% IPCs in the combinatorial group, 3.2172.11% in the inducer group and 0%
in the control Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment
& 2014 International Society of Differentiation Published by Elsevier B.V All rights reserved
1 Introduction
Diabetes mellitus is a highly prevalent disease estimated by the
World Health Organization to affect approximately 500 million
people worldwide However, to date, there is still no cure All of
the current methods used to treat diabetes mellitus aim to restore
glucose homeostasis Cellular therapy has long been considered as
a potential approach to cure this disease However, beta cell numbers are limited, and thus not ideal for replacement therapy Insulin-producing cells (IPCs), on the other hand, can be differ-entiated from stem cells and offer a potential source of cells in lieu
of beta cells For this reason, numerous studies have been conducted to establish protocols to differentiate stem cells into IPCs
Various sources of stem cells have been successfully differen-tiated into IPCs, including embryonic stem cells (Hua et al., 2014; Jiang et al., 2007), induced-pluripotent stem cells (Alipio et al., 2010; Jeon et al., 2012; Zhu et al., 2011), pancreatic stem cells (Noguchi et al., 2010), mesenchymal stem cells from human umbilical cord blood (UCB) (Parekh et al., 2009; Phuc et al.,
2011), placenta (Kadam et al., 2010), bone marrow (Phadnis
et al., 2011), and adipose tissue (Chandra et al., 2009) Of these, UCB-derived MSCs offer several advantages, particularly because
of the increased banking of UCB samples in recent years
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Differentiation
http://dx.doi.org/10.1016/j.diff.2014.08.001
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Abbreviations: DMEM, Dulbecco’s modified eagle medium; GFP, green fluorescent
protein; MNC, mononuclear cell; mRNA, messenger RNA; MSC, mesenchymal stem
cell; IMDM, Iscove’s modified Dulbecco’s media; IPC, insulin producing cell; PBS,
phosphate buffered saline
n Corresponding author.
E-mail addresses: pvphuc@hcmuns.edu.vn (P Van Pham),
Trang 2Consequently, UCB-MSCs have been extensively studied for IPC
differentiation Until now, the most successful methods to induce
UCB-MSC differentiation into IPCs used nicotinamide and/or
exendin-4 inducers (Phuc et al., 2011; Prabakar et al., 2012; Tsai
et al., 2012) Other studies have also successfully differentiated
UCB-MSCs into IPCs by up-regulating some of the master genes
that cause IPC differentiation (mainly PDX-1) (He et al., 2011;
Wang et al., 2011) These studies have demonstrated that PDX-1 is
an important factor regulating pancreatic-endocrine
differentia-tion, particularly for beta cell formation and function
Further-more, PDX-1-differentiated IPCs can regulate the glucose
concentration of diabetic mice
The chemical induction of IPCs from MSCs, however, is
gen-erally poor and, although PDX-1 up-regulation can significantly
increase IPC production, the use of vector viruses, such as an
adenovirus or a lentivirus, harbors the risk of insertional
muta-genesis and immunogenicity (Dave et al., 2009; Hacein-Bey-Abina
et al., 2008; Howe et al., 2008) As such, the differentiated IPCs
from these protocols cannot be used to treat humans in clinical
applications Therefore, this study aimed to develop a novel and
safe method to improve the differentiation efficiency of UCB-MSCs
into IPCs We show improved chemical differentiation of MSCs
following transfection of PDX-1 mRNA
2 Materials and methods
2.1 Isolation of UCB-MSCs
Human UCB was obtained from hospital samples with informed
consent obtained from the mother after delivery of her child All
procedures and manipulations were approved by our Institutional
Ethical Committee (Laboratory of Stem Cell Research and Application,
University of Science, Vietnam National University, Ho Chi Minh City,
Vietnam) and the Hospital Ethical Committee (Nhan Dan 115
Hospital, Ho Chi Minh City, Vietnam) A bag system containing
17 mL of anticoagulant (citrate, phosphate, and dextrose) was used
All UCB units were processed within 3 h after delivery To isolate
mononuclear cells (MNCs), each UCB unit was diluted 1:1 with
phosphate-buffered saline (PBS) and carefully loaded onto
Ficoll-Hypaque (1.077 g/mL, Sigma-Aldrich, St Louis, MO) After density
gradient centrifugation at 3000 rpm for 20 min at room temperature,
MNCs were removed from the interphase, washed twice with PBS,
and resuspended in Iscove’s modified Dulbecco’s media (IMDM)
with 15% fetal bovine serum (FBS) and 1% antibiotic-antimycotic
(Sigma-Aldrich) MNCs were seeded in T-75 cm2 flasks at 1 105
cells/cm2and incubated at 371C, 5% CO2 The medium was replaced
every 3 days When cells reached 70–80% confluence, they were
sub-cultured at a ratio of 1:3 using the same medium as primary culture
2.2 UCB-MSC characterization
UCB-MSCs were characterized according the MSC standard set
by Dominici et al (2006) UCB-MSCs were confirmed by flow
cytometry using surface marker expressions of CD14, CD34, CD45,
HLA-DR, CD73, CD90 and CD105 Flow cytometry was performed
on a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA)
UCB-MSCs were stained with anti-CD14-FITC, anti-CD34-FITC,
anti-CD45-FITC, anti-HLA-DR-FTIC, anti-CD73-PE, anti-CD90-FITC
and anti-CD105-FITC monoclonal antibodies A total of 10,000 cells
were analyzed by CellQuest Pro software Isotype controls were
used in all analyses
UCB-MSCs were also confirmed by their potential to
differenti-ate along multiple lineages Adipogenic differentiation of MSCs
was performed as described previously (Lee et al., 2004b) Briefly,
UCB-MSCs at passage 5 were plated at a density of 1 104
cells/well in 24-well plates At 70% confluence, the cells were switched to IMDM supplemented with 0.5 mM 3-isobutyl-1-methyl-xanthine, 1 nM dexamethasone, 0.1 mM indomethacin and 10% FBS (all from Sigma-Aldrich) and cultured for 21 days Adipogenic differentiation was evaluated by observing the produc-tion of lipid vesicles within cells via microscopy
For osteogenic differentiation, UCB-MSCs were plated at 1 104
cells/well in 24-well plates At 70% confluence, the cells were switched to IMDM supplemented with 10% FBS, 107M dexametha-sone, 50μM ascorbic acid-2 phosphate and 10 mM β-glycerol phosphate (all from Sigma-Aldrich), and cultured for 21 days, as described elsewhere (Lee et al., 2004b) Osteogenic differentiation (calcium accumulation) was confirmed by Alizarin red staining For chondrogenic differentiation, UCB-MSCs were induced using a commercial medium for chondrogenesis (StemPro Chon-drogenesis Differentiation Kit, A10071-01, Life Technologies) UCB-MSCs were differentiated in pellet form, according to manufac-turer’s guidelines After 21 days growth, cell pellets were stained with an anti-aggrecan monoclonal antibody (BD Bioscience) 2.3 In vitro mRNA PDX-1 production
pcDNA3.1-hPDX-1 was amplified by PCR with 50-T7 primer (50-TAATACGACTCACTATAGGG-30) and 30-specific primer for PDX-1 (50-GTCCTCCTCCTTTTTCCAC-30) pcDNA3.1-hPDX-1 was prepared in the previous study by cutting hPDX-1 from vector pWPT-PDX1 with NotI and BamHI (Plasmid 12256, Addgene, Cambridge, MA) and inserting to vector pcDNATM 3.1 (Invitrogen, Carlsbad, CA) (Nguyen
et al., 2014)
The PCR products for hPDX-1 were purified using the GenElute PCR Clean-up Kit, Sigma-Aldrich, St Louis, MO) The purified PCR product was employed for an in vitro transcription reaction using the T7 mScript Standard mRNA Production System (Epicentre Biotechnologies, Madison, WI) The mRNA concentration was measured using a Nanophotometer (Eppendorf, Germany) 2.4 mRNA PDX-1 transfection
UCB-MSCs were transfected according to a previously pub-lished protocol (Arnold et al., 2012) UCB-MSCs were transfected with 3μg of mRNA by nucleofection (NHDF-VPD-1001, Lonza) After transfection, these cells were plated into T-25 flasks and cultured in the medium At 72 h, 144 h, and 216 h after nucleofec-tion, the adherent cells were transfected with “FuGENE HD” (Roche, Basel, Switzerland) according to the manufacturer’s instructions, which was replaced with culture medium 4 h later The ratio of“FuGENE HD” reagent and mRNA was 8μL per 3μg of mRNA Transfected samples of UCB-MSCs were evaluated for changes in Pdx-1 expression at both transcriptional and transla-tional levels
2.5 RNA extraction and reverse transcript real-time RT PCR RNA was extracted from cell cultures using a Trizol extraction kit (Intron Biotechnology, Korea) mRNA was reversed trans-cribed into cDNA using an AMV reverse transcription kit (Agilent Technologies, Santa Clara, CA) The real-time RT-PCR reactions were carried out using Brilliant II SYBRsGreen QPCR Master Mix (Agilent Technologies) The primer sequences were as follows: GAPDH, forward, 50-AGAAGGCTGGGGCTCATTTG-30, and reverse,
50-AGGGGCCATCCACAGTCTTC-30; PDX-1, forward, 50-GGATGAAGTC TACCAAAGCTCACGC-30, and reverse, 50-CCAGATCTTGATGTGTCTC TCGGTC-30; INSULIN, forward, 50-AACCAACACCTGTGCGGCT CA-30; reverse, 50-TGCCTGCGGGCTGCGTCTA-30; NGN3, forward,
50-CGCCGGTAGAAAGGATGAC-30, reverse: 50 -GAGTTGAGGTTGTG-CATTCG-30; NKX6.1, forward: 50-CTGGAGAAGACTTTCGAACAA-30,
Trang 3reverse, 50-AGAGGCTTATTGTAGTCGTCG-30 GAPDH was used as an
internal control for the normalization of gene expression
Real-time RT-PCR was performed as per the following cycling
condi-tions: 951C for 30 s; 60 1C for 30 s; and 72 1C for 60 s for 40 cycles
The Ct values were used to calculate gene expression according to
the 2ΔCCtmethod.
2.6 IPC differentiation
In this study, UCB-MSCs were treated under three conditions:
combinatorial group (PDX-1-transfected and chemically induced);
inducer group (chemically induced only); and control group
(no induction or transfection) At the start of the assay (herein
referred to as day-9 of the time line), undifferentiated UCB-MSCs in
the combinatorial group were transfected with PDX-1 mRNA Nine
days later at‘day 0’, UCB-MSCs in the combinatorial and inducer
groups were induced to differentiate into IPCs in IMDM
supple-mented with 2% FBS, 100 ng/mL epidermal growth factor and 2%
B27 for 3 days From days 4 to 21, cells were incubated in IMDM
supplemented with 10 nM nicotinamide, 2% B27, 10 ng/mL
betacel-lulin and 0.1 mM beta-mercaptoethanol, with fresh media replaced
every 3 days UCB-MSCs in the control group only were cultured
in basic medium (IMDM plus 15% FBS, 1% antibiotic-antimycotic)
At days 7, 14, and 21, the gene expression levels of PDX-1, NGN3,
NKX6.1 and INSULIN were evaluated At day 21, the supernatant
from all three groups was collected to analyze the amount of
insulin production and cell extracts were used to measure
C-peptide production
2.7 Flow cytometry analysis
PDX-1-transfected cells were analyzed byflow cytometry About
5 106 cells were fixed in PFA 4% and permeabilized with 0.03%
Triton X-100 (Sigma-Aldrich, St Louis, MO) diluted in PBS with 0.1%
bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, MO) for 1 h
Non-specific sites were blocked using 10% BSA for an additional 1 h
Cells were then incubated with anti-Pdx1 monoclonal antibody (BD
Bioscience) for 4 h Stained cells were washed three times with PBS
plus 0.1% BSA and then incubated with an anti-mouse secondary
IgG1-FITC for 1 h Finally, the cells were washed three times with PBS
plus 0.1% BSA and re-suspended in FACSfluid sheath for analysis and
sorting on a FACSJazzflow cytometer (BD Bioscience) Isotypes were
used for this analysis
2.8 Immunohistochemistry
For immunohistochemistry, cells werefixed with 4%
paraformal-dehyde and then permeabilized by 0.03% Triton X-100 diluted in PBS
with 0.1% BSA for 1 h Non-specific sites were blocked using 10% BSA
for an additional 1 h Cells were then stained with primary
mono-clonal antibodies against Pdx-1 (sc-390792, Santa Cruz
Biotechnol-ogy, Canada) insulin (sc-52035, Santa Cruz BiotechnolBiotechnol-ogy, Canada)
Following washing with PBS, cells were then stained with
anti-mouse secondary IgG1-FITC (sc-2010, Santa Cruz Biotechnology,
Canada) for 30 min, washed again and then counterstained with
Hoechst 33342 for 10 min (Sigma-Aldrich, St Louis, MO) Staining was
observed under afluorescent microscope (Cell Observer, Carl-Zeiss,
Germany)
2.9 Insulin and C-peptide measurement
After 21 days of differentiation, cells were washed with PBS and
then incubated for 3 h in DMEM-LG (low glucose; 5 mM glucose)
The supernatant was collected and stored at 20 1C for insulin
measurement and the differentiated cells were harvested and the
cellular extract used for C-peptide measurement The Insulin ELISA
kit (ab100578, Abcam, Cambridge, MA) and C-peptide ELISA kit (ab178641, Abcam, Cambridge, MA) were used according to the manufacturer’s instructions The assays were read at 450 nm in a DTX Multimode plate reader (Beckman Coulter, Fullerton, CA) 2.10 Glucose response assay
At day 21 of differentiation, cells were washed with PBS and then incubated for 1 h in DMEM-LG The supernatant was col-lected and stored at 20 1C The cells were washed with PBS and incubated for an additional 1 h in DMEM-HG (high glucose;
25 mM glucose) and this supernatant was also collected and stored at20 1C The insulin concentration was determined using the Insulin ELISA kit, as described above
2.11 Statistical analysis Significance of differences between mean values was assessed
by t test and ANOVA A P value o0.05 was considered to be significant All data were analyzed by Prism 6 software (GraphPad Software, La Jolla, CA)
3 Results 3.1 UCB-MSCs fully exhibited the MSC characteristics
We successfully isolated MSCs from five samples of human UCBs All potential isolates of MSCs were confirmed according to the guidelines set out by Dominici et al (2006) The results showed that isolated MSCs exhibited afibroblast-like shape when cultured under adherent conditions (Fig 1A) These cells were positive for CD44, CD73, and CD90 and negative for CD14 (a marker of monocytes), CD34 (a marker of hematopoietic stem cells), CD45 (a marker of leukocytes) and HLA-DR (Fig 1E–L) The MSCs could also be successfully differentiated along three different mesenchymal lineages, including adipocyte, osteocyte and chon-drocyte lineages For adipogenic differentiation, MSCs demon-strated a change in shape and the presence of lipid droplets within the cytoplasm of cells following growth in adipocyte-inducing medium These lipid droplets were stained red by Oil Red dye (Fig 1B) For osteogenic differentiation, MSCs also demon-strated a change in shape as well as an accumulation in cytoplas-mic calcium, as determined by Alizarin red staining (Fig 1C) Finally, MSCs were also successfully differentiated into chondro-cytes, with pellets staining positively for aggrecan, a specific marker of chondrocytes (Fig 1D)
3.2 PDX-1 mRNA transfection and PDX-1 expression in MSCs
In this experiment, we transfected UCB-MSCs with PDX-1 mRNA according to a published protocol (Arnold et al., 2012)
We found that, compared with un-transfected control cultures (Fig 2A–E), some of the transfected cells showed positive PDX-1 expression, as determined using immunochemistry staining (Fig 2F–K) Using flow cytometry, we determined the PDX-1-positive cell population as 12.5575.32% of the total number of cells (Fig 2L and M)
3.3 Differentiation of UCB-MSCs into IPCs by chemical induction with or without PDX-1 mRNA transfection
To evaluate the efficacy of PDX-1 mRNA transfection on IPC differentiation of UCB-MSCs, we set up two experimental groups and a control group herein referred to as the combinatorial group (Pdx-1-transfected and chemically induced), inducer group
Trang 4(chemically induced only), and control group (no induction or
transfection) In both experimental groups, UCB-MSCs started to
form cell clusters that resembled islets after 14 days of culture
(Fig 3B and E), with no cluster formation observed in the control
group (Fig 3A and D) However, different condensation patterns
were noted between the inducer and combinatorial groups, with
UCB-MSCs in the combinatorial group triggering earlier cell cluster
formation (Fig 3E and F vs.Fig 3B and C) By counting the number
of cell clusters formed in the same time frame between inducer
and combinatorial groups, we found that the number of cell
clusters formed in the combinatorial group (89725 clusters) was significantly higher than that in the inducer group (53731 clusters)
We next assessed the expression of pancreatic cell-related genes
at days 7, 14 and 21 for all three groups Our results showed differences in the expression of Pdx-1, Ngn3, Nkx6.1 and insulin between the groups and between the successive timepoints (Fig 3G and H) After day 7, in comparison with the control group, cells in the inducer group showed 2.4770.9, 1.4370.31, 15.374.9 times higher expression of Pdx-1, Ngn3, Nkx6.1, respectively In the combinatorial
Fig 2 Mesenchymal stem cells (MSCs) expressed PDX-1 after transfection with PDX-1 mRNA MSCs transfected with Pdx-1 mRNA showed expression of PDX-1 protein as detected by immunocytochemistry ((F)–(K)) as compared with untransfected control cells ((A)–(E)) PDX-1-positive cells were analyzed by flow cytometry in the experimental group (M) and the control (L) (Magnification: 100 ).
Fig 1 Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) Isolated cells complied with the minimal standards for defining MSCs: they were adherent with a fibroblast-like shape (A); they were successfully differentiated into adipocytes stained with Oil Red (B), osteoblasts stained with Alizarin Red (C), and chondrocytes stained with anti-aggrecan-PE and Hoechst 33342 (D); they expressed CD44 (H), CD73 (I), CD90 (K); and lacked CD14 (E), CD34 (F), CD45 (G) and HLA-DR (L) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trang 5group, Pdx-1, Ngn3, Nkx6.1 expression was significantly higher than
that in the inducer group (5.9371.5 vs 2.4770.9, 2.7770.70
vs 1.4370.31, 33.0772.76 vs 15.374.9, respectively) At this time
point, insulin was not expressed in UCB-MSCs in the inducer group,
but was present in cells in the combinatorial group (Fig 3G)
After 14 days of induction, UCB-MSCs in both experimental
groups increased the gene expression of Pdx-1, Ngn3, Nkx6.1 and
Insulin, again with higher gene expression observed in the
combi-natorial group as compared with the inducer group (8.6771.86 vs
3.5770.65 for PDX-1; 10.771.31 vs 4.5770.70 for Ngn3;
50.579.8 vs 30.379.8 for Nkx6.1; and 3.8771.5 vs 0.8370.25
for insulin) (Fig 3H) At day 14, insulin expression was now also
detected in the inducer group
After 21 days of induction, there was a reduction in almost all
of the genes, with the exception of insulin, especially in the
combinatorial group Pdx-1, Ngn3, Nkx6.1 expression for the
combinatorial and inducer groups, respectively, were 3.970.1
and 2.2270.99; 2.070.36 and 0.8370.15; and 3.771.1 and
1.5370.7 Insulin expression increased from 3.8771.50 at day
14 to 8.4770.81 at day 21 for the combinatorial group and from
0.8370.25 at day 14 to 2.8770.55 at day 21 for inducer group
(Fig 3I) At this time point, cell cultures were also stained with
anti-insulin monoclonal antibody to determine the translational
expression level of insulin The results showed that cells in
islet-like cell clusters expressed protein insulin whereas cells outside the cell clusters did not (Fig 4)
To determine the differentiation efficiency, we counted the percentage of insulin-positive cells in the experimental and con-trol groups The results showed that there were 8.372.5% (Fig 5E and F) insulin-positive cells in the combinatorial group versus 3.2172.11% in the inducer group (Fig 5C and D) and 0% in the control group (Fig 5A and B)
Differentiated cells at day 21 were induced with two different concentrations of glucose (5 mM and 25 mM) At both concentra-tions of glucose, induced cells in the two experimental groups produced insulin in the supernatant and C-peptide in the cell extract However, the insulin and C-peptide concentrations expressed by cells
in the combinatorial group were higher than those for cells in the inducer group (Fig 5G) With 5 mM glucose, insulin was recorded as 6.76771.747μg/L in the combinatorial group as compared with 2.971.217μg/L in the inducer group and 0.46770.252μg/L in the control group Similarly, the C-peptide concentration reached 30.533711.804 ng/L in the combinatorial group as compared with 12.03373.424 ng/L in the inducer group and 0 ng/L in the control group With 25 mM glucose, the induced cells produced significantly higher amounts of insulin and C-peptide; for example, in the combinatorial group, cells produced 13.8071.389μg/L insulin and 55.900711.758 ng/L C-peptide in 25 mM glucose as compared with
Fig 3 Changes in cell shape and gene expression during differentiation Cells in both inducer (chemical induction only) (B) and combinatorial (Pdx-1 mRNA transfection and chemical induction) (E) groups formed cell clusters after 7 days of induction as compared with cells at Day 0 ((A) and (D), respectively in chemical induction and combinatorial group) These cell clusters were more condensed after 21 days and formed structures that resembled islets ((C) and (F), respectively in chemical induction and combinatorial group) Some of the genes related to beta cells were expressed after 7 days (G), 14 days (H) and 21 days (I) in both experimental groups as compared with the control.
Trang 66.76771.747μg/L and 30.533711.804 ng/L in 5 mM glucose In the
inducer group, cells also increased the amount of insulin and
C-peptide at 25 mM glucose, with 6.56772.723μg/L insulin and
20.56776.100 ng/L C-peptide However, the concentration of insulin
in the supernatant of the control sample was not significantly
affected by glucose (0.43370.153μg/L), and the cells in this group
also showed a complete absence of C-peptide production (Fig 5G
and H)
4 Discussion
An improvement in the differentiation efficiency of UCB-MSCs
into IPCs is an important step if UCB-MSCs are to be used for the
treatment of diabetic mellitus in the clinical setting Most of the
current methods use chemical induction to cause IPC
differentia-tion but with limited success Although previous studies have
successfully reprogrammed UCB-MSCs into pancreatic endocrine
lineage cells, the results held other limitations, including the use of
virus transfection vectors to induce Pdx-1 expression Numerous
previous reports have successfully reprogrammed pluripotent
stem cells using mRNA transfection Thus, we took advantage of
this knowledge to induce UCB-MSC differentiation into IPCs in
combination with chemicals
In thefirst step, we successfully isolated UCB-MSCs that complied
with the suggested guidelines for MSC identification (Dominici et al.,
2006) These cells were positive for CD44, CD73, and CD90 markers
and negative for CD14, CD34, CD45 and HLA-DR, similar to previous
studies (Lee et al., 2004a, 2004b) These cells also were successfully
differentiated into adipocytes, osteoblasts, and chondrocytes and
thus determined to be MSCs for the subsequent experiments
We next applied PDX-1 mRNA transfection system to improve
differentiation of MSCs into IPCs in combination with traditional
inducers such as nicotinamide, among others Pdx-1, also known as
insulin promoter factor 1, is a transcription factor for pancreatic
development and beta cell maturation PDX-1 has been used
previously as a reprogramming factor to trigger the differentiation
of MSCs from various sources– bone marrow (Guo et al., 2012; Li
et al., 2007; Limbert et al., 2011; Sun et al., 2006; Zaldumbide et al.,
2012), UCB (He et al., 2011; Wang et al., 2011), and adipose tissue
(Boroujeni and Aleyasin, 2013) – into endocrine pancreatic cells
Pdx-1 was thus confirmed as an important factor for MSC
differ-entiation However, in most of the previous studies, Pdx1 is delivered
to the target cells via virus-based vectors, such as adenoviruses (Guo
et al., 2012; He et al., 2011; Li et al., 2007; Zaldumbide et al., 2012) or
retroviruses (Boroujeni and Aleyasin, 2013; Limbert et al., 2011;
Rahmati et al., 2013; Talebi et al., 2012) These viral vectors hold
some inherent risks such as insertional mutagenesis and/or vector mobilization following viral infection (Dave et al., 2009; Hacein-Bey-Abina et al., 2008; Howe et al., 2008), which limit their use in the clinical setting The recent study byBoroujeni and Aleyasin (2013) used a non-integrated lentiviral vector carrying Pdx-1 to induce the adipose derived stem cells (ADSCs) into IPCs, which provided a new safe method for transient transfection However, the procedure for using a non-integrated lentiviral vector is complex, difficult and time consuming
RNA-based reprogramming tools are considered to better instigate cellular reprogramming for clinical applications (Bernal,
2013) mRNA is one of best RNA-based tools that has been proven
to be safe, particularly in terms of its lack of immunogenicity (Bernal, 2013) Moreover, mRNA transfection can be used to
efficiently reprogram some cells Indeed, some authors have successfully activated the pluripotency of fibroblasts by mRNA (Mandal and Rossi, 2013; Plews et al., 2010; Tavernier et al., 2012; Warren et al., 2010) In an earlier study, Wiehe et al (2007) demonstrated that mRNA could efficiently promote protein expression of DeltaLNGFR in human hematopoietic stem cells and MSCs by nucleofection with DeltaLNGFR mRNA (Wiehe
et al., 2007) Immature dendritic cells (DCs) can also be forced to mature into antigen-presenting cells by electroporation of mRNAs encoding a tumor antigen, CD40 ligand, CD70 and constitutively active (caTLR4) (Van Nuffel et al., 2010) In this way, DC vaccines are used safely in cancer treatment, for instance, to induce antigen-specific T-cell responses in melanoma patients (Aarntzen
et al., 2012; Bonehill et al., 2009; Van Nuffel et al., 2012; Wilgenhof
et al., 2011), ovarian carcinoma and carcinosarcoma patients and (Coosemans et al., 2013), leukemia patients (Overes et al.,
2009) mRNA has also been used to re-direct stem cell fate In a recent study, the authors directly injected VEGF mRNA into a myocardial site, and found a marked improvement in heart function because of the mobilization of epicardial progenitor cells and their redirection toward to cardiovascular cells (Zangi et al.,
2013) Lui et al (2013)were also able to drive the multipotent Isl1þ heart progenitors into endothelial cells by VEGF mRNA (Lui
et al., 2013)
Thus, it is with this supportive literature that we chose to use the mRNA-based tool to express the transcription factor PDX-1 in UCB-MSCs in this study Because the transgenic system is based on mRNA, this technique is a safe approach for generating clinically relevant IPCs We were uncertain, however, whether PDX-1 mRNA would be able to cross the phospholipid membrane to the cyto-plasm Indeed, ourfindings indicate that PDX-1 mRNA can cross the membrane, even though the ratio of PDX-1-positive cells detected
byflow cytometry and immunocytochemistry were low Taking into
Fig 4 Insulin synthesis from islet-like cell clusters after differentiation Cell clusters were positive with anti-insulin monoclonal antibody (FITC, green) Cells in the cell cluster expressed insulin in both combinatorial (Pdx-1 mRNA transfection and chemical induction) ((A)–(E)) and inducer (chemical induction only) ((F)–(K)) groups, whereas cells outside the islets were negative for insulin Cells were counterstained with Hoechst 33342 (blue color) (Magnification: 100 ) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trang 7consideration that different mRNAs have different capacities to
cross the cell membrane, we believe that a higher number of cells
received the mRNA than expressed the protein, with only some cells
receiving sufficient PDX-1 mRNA to express PDX-1 protein in high
enough concentrations for detection by the naked eye as compared
withflow cytometry and immunocytochemistry Indeed, all tools
that are not based on DNA integration can only be effective when
proteins are translated These proteins are active components that
trigger cell fate, and our findings show that PDX-1 expression triggering the differentiation of UCB-MSCs into endocrine pancrea-tic cells Pdx-1 has been shown to regulate a number of important genes that dictate pancreas formation and differentiation as well as beta cell function: insulin (Ohlsson et al., 1993), glucose transporter
2 (Glut2) (Waeber et al., 1996), glucokinase (Watada et al., 1996) and islet amyloid polypeptide (Bretherton-Watt et al., 1996; Carty et al., 1997; Serup et al., 1996)
Fig 5 Differentiated cell cultures contained insulin-positive cell populations and exhibited glucose-dependent responses Flow cytometry showed that the percentage
of insulin-positive cells in the combinatorial (Pdx-1 mRNA transfection and chemical induction) group ((E) and (F)) was higher than that in the inducer (chemical induction only) group ((C) and (D)) There were 0% insulin-positive cells in the control group ((A) and (B)) Concentrations of insulin (from supernatant) and C-peptide (from cell extract) in the combinatorial group were higher than those in the inducer group with both 5 mM and 25 mM glucose (G).
Trang 8We showed that the combinatorial group was able to signi
fi-cantly increase the expression of genes related to beta cells over
cells subjected to chemical induction alone, demonstrating the
utility of PDX-1 mRNA transfection The results illustrate that
although the chemical inducers have a positive effect on
pancrea-tic differentiation, the active expression of PDX-1 helps to facilitate
and accelerate this process The results from the gene analysis
showed that PDX-1 expression initiated this process In the control
group, UCB-MSCs expressed extremely low levels of PDX-1 and
thus were unable to differentiate into IPCs In contrast, UCB-MSCs
expressed Pdx-1 mRNA at the transcriptional level in both
experi-mental groups By quantitative analysis, PDX-1 mRNA expression
in the combinatorial group was higher than the endogenous
expression of PDX-1 identified in cells in the inducer group This
difference in the expression level of PDX-1 between these two
groups subsequently caused a difference in the gene expression
levels of Ngn3, Nkx6.1, and insulin In addition, the difference in
insulin transcription because of these levels of PDX-1 affected the
C-peptide concentration These results were similar to those of
another previously published study (Yuan et al., 2012).Yuan et al
(2012)showed that the expression of Pdx-1 correlated with the
level of insulin at both the transcriptional and translational levels
(Yuan et al., 2012) Our results from the analysis of insulin-positive
cells after 21 days of induction also indicated that the percentage
of insulin-positive cells (8.372.5%) was higher than cultures
subjected to chemical induction only Thisfinding was similar to
that reported for human embryonic stem cell differentiation into
IPCs (Jiang et al., 2007) Interestingly, our insulin production levels
were higher than those achieved by transfecting bone
marrow-derived mesenchymal stem cells with plasmid vectors containing
Pdx-1 and Betacellulin, with only5% insulin-positive cells in that
study (Li et al., 2008); this was similar to the transduction of PDX-1
in UCB-MSCs with an adenovirus vector (11.6174.83%
insulin-positive cells) (Wang et al., 2011) However, all of these results,
including our own study, showed a lower expression of insulin as
compared with PDX-1 transduction by lentiviral vector (28.23%
insulin-positive cells) (Sun et al., 2006)
Although we also detected some insulin in the control group,
we did not detect C-peptide in this group It is thus possible that
the insulin detected in this group could be from cross-reaction
between bovine insulin in the FBS This study showed that PDX-1
mRNA transfection could not only increase the percentage of IPCs
but also produce functional IPCs IPCs in both experimental groups
could produce insulin in a glucose-dependent manner Thus,
PDX-1 mRNA transfection increased the differentiation efficiency
with-out interfering with the normal pancreatic differentiation process
However, the level of C-peptide as well as insulin produced from
IPCs had not been compared to them in islets of Langerhans
Another limitation was insulin production of IPCs had not been
evaluated for a long time Quality of IPCs needs to be evaluated
before they can be used in preclinical and clinical trials
5 Conclusions
The establishment of an efficient and safe protocol for
UCB-MSC differentiation into IPCs is a crucial step for the utility of UCB-MSCs
in the treatment of clinical diabetes mellitus This study showed
that PDX-1 mRNA transfection in combination with chemical
inducers is a safe and efficient method to improve UCB-MSC
differentiation into IPCs PDX-1 mRNA transfection significantly
increased the percentage of IPCs in the differentiated cell
popula-tion as compared with the use of chemical inducpopula-tion alone These
differentiated cells strongly expressed some of the genes related to
beta cell function and produced insulin and C-peptide in a
glucose-dependent manner These results provide a new method for the potential clinical application of IPCs from UCB-MSCs
Authors’ contributions PVP carried out studies including primary culture of MSCs, MSC transfection, flow cytometry analysis, gene analysis PTMN and ATQN collected umbilical cord blood; prepared and isolation of plasmid containing Pdx-1 gene; PCR product purification VMP, ANTB, LTTD, KGN take care MSCs before and after transfection, ELISA analysis, PCR preparation NKP revised the manuscript, spelling and grammaticalfixation
Competing interests The authors declare that they have no competing interests
Acknowledgements This work was funded by grants from Vietnam National Uni-versity, Ho Chi Minh city, Vietnam (B2010-18-02TD), and Ministry
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