Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Contents lists available at ScienceDirect Differentiation journal homepage: www.elsevier.com/locate/diff Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Phuc Van Pham n, Phuoc Thi-My Nguyen, Anh Thai-Quynh Nguyen, Vuong Minh Pham, 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 art ic l e i nf o a b s t r a c t Article history: Received 29 April 2014 Received in revised form August 2014 Accepted 18 August 2014 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 8.3 2.5% IPCs in the combinatorial group, 3.21 72.11% 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 Keywords: Mesenchymal stem cells UCB-MSCs Insulin producing cells PDX-1 mRNA transfection 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 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), ntmphuoc@hcmus.edu.vn (P Thi-My Nguyen), nguyenthaiquynhanh@gmail.com (A Thai-Quynh Nguyen), pmvuong@hcmus.edu.vn (V Minh Pham), bntanh@hcmus.edu.vn (A Nguyen-Tu Bui), dttloan@hcmus.edu.vn (L Thi-Tung Dang), ngkhue@hcmus.edu.vn (K Gia Nguyen), pkngoc@hcmus.edu.vn (N Kim Phan) 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 differentiated 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 differentiated 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 http://dx.doi.org/10.1016/j.diff.2014.08.001 Join the International Society for Differentiation (www.isdifferentiation.org) 0301-4681/& 2014 International Society of Differentiation Published by Elsevier B.V All rights reserved Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Consequently, 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 differentiation, particularly for beta cell formation and function Furthermore, PDX-1-differentiated IPCs can regulate the glucose concentration of diabetic mice The chemical induction of IPCs from MSCs, however, is generally 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 mutagenesis 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 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 h after delivery To isolate mononuclear cells (MNCs), each UCB unit was diluted 1:1 with phosphate-buffered saline (PBS) and carefully loaded onto FicollHypaque (1.077 g/mL, Sigma-Aldrich, St Louis, MO) After density gradient centrifugation at 3000 rpm for 20 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  105 cells/cm2 and incubated at 37 1C, 5% CO2 The medium was replaced every days When cells reached 70–80% confluence, they were subcultured at a ratio of 1:3 using the same medium as primary culture cells/well in 24-well plates At 70% confluence, the cells were switched to IMDM supplemented with 0.5 mM 3-isobutyl-1methyl-xanthine, 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 production of lipid vesicles within cells via microscopy For osteogenic differentiation, UCB-MSCs were plated at  104 cells/well in 24-well plates At 70% confluence, the cells were switched to IMDM supplemented with 10% FBS, 10 À M dexamethasone, 50 μM 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 Chondrogenesis Differentiation Kit, A10071-01, Life Technologies) UCBMSCs were differentiated in pellet form, according to manufacturer’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 published protocol (Arnold et al., 2012) UCB-MSCs were transfected with μg 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 nucleofection, the adherent cells were transfected with “FuGENE HD” (Roche, Basel, Switzerland) according to the manufacturer’s instructions, which was replaced with culture medium h later The ratio of “FuGENE HD” reagent and mRNA was μL per μg of mRNA Transfected samples of UCB-MSCs were evaluated for changes in Pdx-1 expression at both transcriptional and translational levels 2.2 UCB-MSC characterization 2.5 RNA extraction and reverse transcript real-time RT PCR 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 differentiate along multiple lineages Adipogenic differentiation of MSCs was performed as described previously (Lee et al., 2004b) Briefly, UCB-MSCs at passage were plated at a density of  104 RNA was extracted from cell cultures using a Trizol extraction kit (Intron Biotechnology, Korea) mRNA was reversed transcribed 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 SYBRs Green 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 -GAGTTGAGGTTGTGCATTCG-30 ; NKX6.1, forward: 50 -CTGGAGAAGACTTTCGAACAA-30 , Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ reverse, 50 -AGAGGCTTATTGTAGTCGTCG-30 GAPDH was used as an internal control for the normalization of gene expression Realtime RT-PCR was performed as per the following cycling conditions: 95 1C 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 Δ the À CCt method 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) 2.6 IPC differentiation At day 21 of differentiation, cells were washed with PBS and then incubated for h in DMEM-LG The supernatant was collected and stored at À20 1C The cells were washed with PBS and incubated for an additional h in DMEM-HG (high glucose; 25 mM glucose) and this supernatant was also collected and stored at À 20 1C The insulin concentration was determined using the Insulin ELISA kit, as described above 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 supplemented with 2% FBS, 100 ng/mL epidermal growth factor and 2% B27 for days From days to 21, cells were incubated in IMDM supplemented with 10 nM nicotinamide, 2% B27, 10 ng/mL betacellulin and 0.1 mM beta-mercaptoethanol, with fresh media replaced every 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 Cpeptide production 2.7 Flow cytometry analysis PDX-1-transfected cells were analyzed by flow cytometry About  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 h Non-specific sites were blocked using 10% BSA for an additional h Cells were then incubated with anti-Pdx1 monoclonal antibody (BD Bioscience) for h Stained cells were washed three times with PBS plus 0.1% BSA and then incubated with an anti-mouse secondary IgG1-FITC for h Finally, the cells were washed three times with PBS plus 0.1% BSA and re-suspended in FACS fluid sheath for analysis and sorting on a FACSJazz flow cytometer (BD Bioscience) Isotypes were used for this analysis 2.8 Immunohistochemistry For immunohistochemistry, cells were fixed with 4% paraformaldehyde and then permeabilized by 0.03% Triton X-100 diluted in PBS with 0.1% BSA for h Non-specific sites were blocked using 10% BSA for an additional h Cells were then stained with primary monoclonal antibodies against Pdx-1 (sc-390792, Santa Cruz Biotechnology, Canada) insulin (sc-52035, Santa Cruz Biotechnology, Canada) Following washing with PBS, cells were then stained with antimouse secondary IgG1-FITC (sc-2010, Santa Cruz Biotechnology, Canada) for 30 min, washed again and then counterstained with Hoechst 33342 for 10 (Sigma-Aldrich, St Louis, MO) Staining was observed under a fluorescent 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 h in DMEM-LG (low glucose; mM glucose) The supernatant was collected and stored at À 20 1C for insulin measurement and the differentiated cells were harvested and the cellular extract used for C-peptide measurement The Insulin ELISA 2.10 Glucose response assay 2.11 Statistical analysis Significance of differences between mean values was assessed by t test and ANOVA A P value o 0.05 was considered to be significant All data were analyzed by Prism software (GraphPad Software, La Jolla, CA) 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 a fibroblast-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 chondrocyte lineages For adipogenic differentiation, MSCs demonstrated a change in shape and the presence of lipid droplets within the cytoplasm of cells following growth in adipocyteinducing medium These lipid droplets were stained red by Oil Red dye (Fig 1B) For osteogenic differentiation, MSCs also demonstrated a change in shape as well as an accumulation in cytoplasmic calcium, as determined by Alizarin red staining (Fig 1C) Finally, MSCs were also successfully differentiated into chondrocytes, 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-1positive cell population as 12.55 75.32% 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 Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Fig 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.) Fig 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  ) (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 (89 725 clusters) was significantly higher than that in the inducer group (53 31 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 Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Fig 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 days of induction as compared with cells at Day ((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 days (G), 14 days (H) and 21 days (I) in both experimental groups as compared with the control group, 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 combinatorial group as compared with the inducer group (8.6771.86 vs 3.57 0.65 for PDX-1; 10.7 1.31 vs 4.577 0.70 for Ngn3; 50.5 79.8 vs 30.3 79.8 for Nkx6.1; and 3.87 1.5 vs 0.83 0.25 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 3.9 70.1 and 2.22 70.99; 2.0 0.36 and 0.83 70.15; and 3.7 71.1 and 1.53 70.7 Insulin expression increased from 3.87 71.50 at day 14 to 8.47 70.81 at day 21 for the combinatorial group and from 0.83 70.25 at day 14 to 2.87 70.55 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 control groups The results showed that there were 8.3 72.5% (Fig 5E and F) insulin-positive cells in the combinatorial group versus 3.217 2.11% 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 concentrations 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 mM glucose, insulin was recorded as 6.76771.747 μg/L in the combinatorial group as compared with 2.971.217 μg/L in the inducer group and 0.46770.252 μg/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 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 13.8071.389 μg/L insulin and 55.900711.758 ng/L C-peptide in 25 mM glucose as compared with Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Fig 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.) 6.76771.747 μg/L and 30.533711.804 ng/L in mM glucose In the inducer group, cells also increased the amount of insulin and C-peptide at 25 mM glucose, with 6.56772.723 μg/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 (0.43370.153 μg/L), and the cells in this group also showed a complete absence of C-peptide production (Fig 5G and H) 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 differentiation 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 the first 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 differentiation 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-BeyAbina et al., 2008; Howe et al., 2008), which limit their use in the clinical setting The recent study by Boroujeni 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 Isl1ỵ 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 UCBMSCs 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 cytoplasm Indeed, our findings indicate that PDX-1 mRNA can cross the membrane, even though the ratio of PDX-1-positive cells detected by flow cytometry and immunocytochemistry were low Taking into Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Fig 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 mM and 25 mM glucose (G) consideration 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 with flow 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 pancreatic 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 (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) Please cite this article as: Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i P Van Pham et al / Differentiation ∎ (∎∎∎∎) ∎∎∎–∎∎∎ We showed that the combinatorial group was able to significantly 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 pancreatic 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 experimental 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 (8.3 72.5%) was higher than cultures subjected to chemical induction only This finding 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 marrowderived mesenchymal stem cells with plasmid vectors containing Pdx-1 and Betacellulin, with only $ 5% 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 (11.61 74.83% insulinpositive 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, PDX1 mRNA transfection increased the differentiation efficiency without 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 Conclusions The establishment of an efficient and safe protocol for UCBMSC differentiation into IPCs is a crucial step for the utility of 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 population as compared with the use of chemical induction 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 grammatical fixation Competing interests The authors declare that they have no competing interests Acknowledgements This work was funded by grants from Vietnam National University, 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mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i ... Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i... Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i... Van Pham, P., et al., Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection Differentiation (2014), http://dx.doi.org/10.1016/j.diff.2014.08.001i