miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.


INTRODUCTION
The pancreatic islets have around a 70% beta cell content [1]. These cells secrete insulin, a hormone of great importance in regulating carbohydrate, fat and protein metabolism. Death and/or dysfunction of beta cells leads to an inadequate amount of insulin, and this in turn gives rise to diabetes mellitus [2]. One of the most common chronic diseases in children and adolescents is type 1 diabetes mellitus, which is an autoimmune destruction of insulin production. Sufferers become dependent on insulin injection, but an exogenous supply of insulin results in severe hypoglycemia-related complications [3,4]. Whole pancreas transplantation restores endogenous insulin production, but perioperative morbidity due to the release of digestive enzymes from the exocrine pancreas during surgery and long-term immunosuppressive drug consumption are major issues [5][6][7][8]. Transplantation of functional pancreatic islet cells is a subject of considerable interest as an alternative to whole pancreas transplantation. In this method, an infusion of fresh donor islets is followed by long-term non-steroidal immunosuppressive drug consumption. The obstacles are the worldwide shortage of insulin-producing islets, immune rejection of islets, and the increased risk of malignancies and opportunistic infections [9,10]. It is essential to find renewable islet replacement tissue sources that do not elicit ethical concerns that impede their clinical application. Postnatal-derived mesenchymal stem cells (MSCs), such as those from adipose tissue, present fewer ethical concerns and are considered possible sources of transplantable insulin-producing cells [5,6,9,11,12]. Due to its plasticity and easy availability, placental tissue is very promising in regenerative medicine. Human placenta-derived mesenchymal stem cells (hPDMSCs) have broad developmental potential, and could be induced to differentiate into insulinproducing cells.
In recent years, one of the most fascinating discoveries in biology was the identification of the family of small, non-coding RNAs known as microRNAs (miRNAs). Initially described in worms [13], microRNAs (miRNAs) regulate the expression of genes through post-transcriptional mechanisms [14]. Recent evidence has revealed that miRNAs play a key role in the control of biological activities such as embryonic development, cell proliferation, metabolic homeostasis and apoptosis [15]. MicroRNAs [16,17] play a direct role in pancreatic islet development [18], beta cell differentiation [19], insulin secretion [20], and the control of glucose homeostasis [21,22]. miRNAs are also involved in secondary complications of diabetes [23,24]. Kloosterman et al. reported that miR-375 plays an important role in pancreatic islet development in zebra fish [18]. A reduction in miR-375 expression was found to cause defects in pancreatic islet morphology. In situ hybridization studies revealed that miR-375 is expressed in endodermal endocrine progenitor cells [25]. The expression of miR-375 is regulated by transcription factors that are important in the development and function of the pancreas, such as hepatocyte nuclear factor 6 (HNF6), neurogenin 3 (Ngn3), neuronal differentiation 1 (NEUROD1), and pancreatic and duodenal homeobox 1 (PDX-1) [26]. Overexpression of miR-375 inhibits insulin secretion. Therefore, miR-375 regulates glucose-stimulated insulin secretion in a negative manner. In the same way, insulin secretion is enhanced by inhibition of endogenous miR-375 using antagomiRs [20]. These data suggest that miR-375 has a dual function of mediating pancreatic islet development and insulin secretion. The objective of this study was to transfect hPDMSCs with miR-375 in order to obtain effective insulin-producing clusters (IPCs) in a short period of time.

Generation of MSCs from human placental decidua basalis (PDB-MSCs)
Full term (gestation ≥ 37 weeks) normal human placentas (n = 2) from cesarean section deliveries were obtained from the obstetrics department of Hafez Hospital, which is affiliated with Shiraz University of Medical Sciences. Informed consent was obtained from the mothers, and the ethical committee of Shiraz University of Medical Sciences approved the study. Infectious pathology was excluded using HIV, HCV and HBV tests. The samples were transported in cold PBS. Initially, a small fragment of the PDB-MSCs was separated, mechanically fragmented, and the resulting pieces were washed with PBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Then, the tissue was minced into small fragments (1-2 mm 3 ). The tissue pieces were transferred to 10-cm 2 plates containing DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, and incubated at 37ºC in a humidified atmosphere containing 5% CO 2 . The explants were left undisturbed for 10 days to allow the migration of cells from the margins of the explants. Upon reaching 70 to 80% confluence, adherent PDB-MSCs were harvested with 0.05% trypsin-EDTA (Gibco), and single-cell suspensions were used for subsequent experiments. Real-time qPCR was used to study the presence of transcription factors that regulate the maintenance of the pluripotent state in ESC (OCT4, Nanog).

Optimization of chemical transfection
The PDB-MSCs were transfected with FAM-Labeled Pre-miR (Ambion) using siPORT NeoFX Transfection Agent (Invitrogen) as recommended in the manufacturer's protocol (Tables 1 and 2). Various cell numbers and FAM concentrations were used. The cells were cultured in DMEM F12, 10% FBS for 24 h and incubated at 37ºC in a humidified atmosphere containing 5% CO 2 , and then 24 and 48 h after transfection, the percentage of fluorescently labeled cells were calculated by flow cytometry (Becton Dickinson). In another experiment, to improve the protocol outcome, cells from the previous experiment were cultured for 72 h and then transfected again. The percentage of fluorescently labeled cells was calculated again by flow cytometry after 24 h. In the final experiment, the percentage of FAM-labeled cells was measured after 96 h and then compared with the results of previous experiments.  Table 3). Then the cells were cultured in 24-well plates containing DMEM/F12 supplemented with 10% fetal bovine serum, penicillin 100 U/ml, streptomycin 100 μg/ml and incubated at 37ºC in a humidified atmosphere containing 5% CO 2 . After 72 h, the cells were transfected again and cultured. FAM-Labeled Pre-miR Negative Control (Ambion) is a non-targeting negative control. It was used at a final concentration of 80 pmol. In this study it was used as a negative control in order to monitor transfection efficiency during the optimization of transfection conditions.

Real-time qPCR or quantification of pancreas-specific transcription factors
Total RNA was extracted from transfected PDB-MSCs (4 and 7 days after transfection) using Mini-RNease RNA extraction kit (Cinnagen). The amount of extracted RNA was measured by OD 260/280 measurement. The cDNA was synthesized using MMULV reverse transcriptase (Cinnagen) from 1 µg of total RNA. The synthesized cDNA was used to examine changes in the levels of pancreas-specific transcription factors from differentiated cells. PCR amplifications were carried out using SYBR Green (PARS Genome) following the manufacturer's instructions. The cDNA synthesized for pancreas-specific transcription factors were normalized to beta-actin. Amplification was as follows: denaturing at 95ºC for 5 min; followed by 40 cycles of denaturation at 95ºC for 1 min; and annealing for 1 min. Any amplification of more than 35 cycles (Ct) was considered negative. The final stage was run to generate a melting curve for verification of the amplification product specificity. The relative expression levels of the genes were calculated according to the formula: 2 _DDCt . After cDNA synthesis, serial dilution (1/10) was prepared and real-time qPCR was done for both the internal control (beta-actin) and the gene of interest (insulin). The efficiency was about 95%, with a slope of -3.43. The primers, conditions and product sizes are summarized in Table 4.

Immunocytochemistry (ICC)
Six days after transfection, the cells were collected, washed with PBS, fixed with acetone for 5 min, and incubated with anti-neurogenin 3 antibody (3B5; Abcam; dilution 1:1000) and anti-insulin (DAKO) overnight. After washing in PBS, the Envision Detection System (DAKO) was used. The slides were washed with PBS for 10 min, visualized with diaminobenzidine (DAB; DAKO) and counterstained with hematoxylin (Sigma-Aldrich). The morphology of the cells was observed under a light microscope. FAM-Labeled Pre-miR Negative Control with no morphological change was used in parallel, in which no staining was observed in ICC.

DTZ staining
Diphenylthiocarbazone (Dithizone, DTZ; Sigma) is a zinc-chelating chemical substance that is used for the identification of IPCs. The dye selectively stains the live pancreatic beta-cells as bright crimson red.

C-peptide and insulin secretion
The adherent clusters produced through this procedure were rinsed twice in The insulin and C-peptide levels were determined using a radioimmunoassay kit (Immunotech) and the results were compared with those for the control group (untransfected MSC cells).

Statistical analysis
The data were presented as means ± SD. Each experiment was repeated 3 times. Data from related assays were assessed by one-way ANOVA followed by Tukey's test for pair wise comparison. p < 0.05 was considered statistically significant. The statistical analyses and design of the graph were performed using Graph Pad Prism 5 software.

Morphological changes before and after transfection with miR-375 in PDB-MSCs
PDB-MSCs formed a homogenous monolayer of adherent and spindle-shaped fibroblast-like cells. In passage 3 (P3), the cells were transfected with has-miR375. After three days, the islet-like cluster was partially formed and MSCs and IPCs appeared on day 6 ( Fig. 1A-D). The induced MSCs shrank after three days and became round in shape. After 6 days, cell clusters were formed. On day 7, the clusters gradually detached from the bottom of the plate and floated in the medium. The PDB-MSCs that were transfected with FAM-Labeled Pre-miR (negative control) did not show any morphological change after 7 days.

Flow cytometry analyses of PDB-MSCs
Flow cytometry analyses showed that the PDB-MSCs were a homogeneous cell population in which more than 90% of the cells were positive for mesenchymal markers (CD90, CD44, CD105), and negative for CD34 and CD133 (Fig. 2), which is in line with the internationally recognized criteria for MSCs [27,28]. Real-time qPCR showed that PDB-MSCs express the embryonic stem cell (ESC) transcriptional factors Nanog and OCT4.

Expression of has-miR-375 in PDB-MSCs
Real-time qPCR was used to determine the expression of has-miR-375. The level of miR-375 increased 3981.23-fold in PDB-MSCs 24 h after chemical transfection.

The effect of has-miR-375 on PDB-MSCs-derived IPCs
Pancreatic islets have high levels of zinc compared with other tissues, so DTZ stains them crimson red [29]. On day 7, the clusters appeared red, suggesting the presence of IPCs (Fig. 4). We examined the expression of pancreatic-specific transcription factors in IPCs using real-time qPCR on days 4 and 7 after chemical transfection (Fig. 5). The character of the differentiated cells was similar to that of pancreatic islet cell development in vivo, but all of the changes happened in a short period.  The expressions of PDX1 and NKX6.1 (specific markers of pancreatic progenitors) increased on day 4 and peaked on day 7, when the expression level of OCT4 had rapidly reduced (Diagram 1). At the same time, the expression levels of NKX6.1, KIR6.2 and PAX4 had increased. The upregulated gene expression of NGN3, PDX1 and GLUT2 was in favor of pancreatic specialization. The expression of insulin increased on day 7, indicating that the progenitors had differentiated towards insulin-producing cells. This protocol also induced the expression of glucagon. The clusters were positive for neurogenin 3 (nuclear and cytoplasmic; Fig. 6A) and insulin (cytoplasmic) protein (Fig. 6B). and glucagon genes increased significantly on day 7. F, H -The expression NGN3 and insulin increased on day 7. These data showed that on day 7, clusters had formed and the cells had ability to express pancreatic-specific transcription factors. The data are shown as means ± SD from three independent experiments. Analysis was performed by one-way ANOVA followed by Tukey's test.

Target genes of miR-375 in the beta-cell differentiation
The target genes of miR-7 were investigated using Mir walk, miRanda (miRBase) and TargetScanS. MiR-375 has ten target genes, and some of them are critical for pancreas development, for example Hnf1β, Sox17, GATA6 and Pax6 [30].  . The bars represent means ± SD for three independent experiments (n = 3). ***P < 0.0001 -significantly different from the negative control group and from each other. Analysis was performed by one-way ANOVA followed by Tukey's test. B -C-peptide release in response to 2.8 and 16.7 mM glucose. On day 6, the glucose-stimulated C-peptide release of the ILCs was measured after 60 min incubation in a different glucose concentration. The result was compared with the negative control group (FAM-Labeled Pre-miR transfected cells). The bars represent the means ± SD for three independent experiments (n = 3). ***P < 0.0001 -significantly different from the negative control group and also from each other. Analysis was performed by one-way ANOVA followed by Tukey's test.

DISCUSSION
During the progression of diabetes, pancreatic beta cells are either destroyed or become dysfunctional [2]. Stem cell therapy is promising for the treatment of diabetic patients and PDB-MSCs are good candidates for cell therapy. These cells are abundant and are an available source with low immunological reactions. According to immune regulatory properties, the cells are also used for overcoming graft-versus-host disease (GVHD) in allogeneic transplantation [31,32].
In the literature, there are many approaches that have been used to induce MSC differentiation into IPCs: various growth factors [33]; chemical substances [34,35] and transfection with viral vectors [36]. Kadam et al. [34] studied the differentiation of human placenta-derived mesenchymal stem cells (hPDMSCs) into insulin-producing cells. Their method had 3 stages. The medium culture in stage 1 contained α-MEM, BSA Cohn fraction V, fatty acid-free insulintransferrin-selenium (ITS). In stage 2, it contained α-MEM, BSA, ITS and taurine. In the final stage, it contained α-MEM, BSA, ITS, taurine, glucagonlike peptide (GLP)-1 and nicotinamide. Talebi et al. [36] studied the differentiation of MSCs from the bone marrow of Sprague-Dawley rats toward islet-like cells using PDX1-transducing lentiviruses. The cells were transplanted to diabetic rats and a reduction in blood glucose level from high to normal was revealed.
However, many problems remain in the differentiation pathway. The differentiation efficiency was low and the differentiated cells were not functional for a long time [36]. Therefore, the main goal of our study was to produce functional IPCs in a short time using a new approach. miRNAs are thought to have important roles in stem cell differentiation [37,38].
One of the first miRNAs to be detected in the pancreas was miR-375 [18]. The expression level of mirR-375 increases during pancreas organogenesis [18,[39][40][41]. Joglekar et al. revealed that the miRNA levels of miR-7, miR-9, miR-375 and miR-376 significantly increased during human pancreatic islet development [42]. Wei et al. induced hES cells into insulin-producing cells and the dynamic expression of microRNAs during the differentiation was evaluated. They found that miR-375 expression increased from day 4, the maximum expression was detected on day 8, and this then decreased until the end of the differentiation [30]. In the Wei et al. study, the maximum expression of miR-375 occurred at the same time that the expression levels of pancreatic hormone encoding genes began to rise [43].
To explore the direct effect of miR-375 on MSC differentiation into IPCs, PDB-MSCs were transfected with miR-375 in our project. Comparison between the increased expression of miR-375 and changes in crucial beta-cell transcription factors, such as insulin, glucagon, PAX4, NGN3, GLUT2, NKX6.1, KIR6.2, PDX1, and GLUT2, demonstrated that miR-375 could promote MSC differentiation toward IPCs. Overall, the expression dynamics during in vitro differentiation of IPCs from PDB-MSCs is similar to that of pancreatic beta-cells development in vivo, but it occurs in a shorter time [44,45]. On day 6, IPCs have the capacity of C-peptide and insulin secretion in response to glucose stimulation. However, our study has a few limitations. The presence of the insulin hormone was confirmed in ICC. This result should be confirmed by immunostaing with human c-peptide antibody. The qPCR results suggest a high induction of glucagon expression. It is necessary to verify if the expression of glucagon co-localizes with insulin, as reported for immature beta cells (polyhormonal cells). Making use of this approach, it is possible to generate functional IPCs in a shorter time than has been seen in other studies. Although overexpression of miR-375 induces MSC differentiation into IPCs, cell differentiation has a very complex network, and microRNAs considered as regulatory factors take part in complex cell regulation systems. Therefore, clarifying the mechanism of MSC differentiation into IPCs could improve the induction efficiency. Further investigation is required.