Induced Hyperglycemia in Mice is Controlled Following the Micro uidic System- Assisted Transplantation of Stem Cells-derived Insulin- producing Cells Transduced with miRNA

Adele Soltani Tarbiat Modares University Faculty of Medical Sciences Masoud Soleimani Tarbiat Modares University Faculty of Medical Sciences Mohammad Adel Ghiass Tarbiat Modares University Faculty of Medical Sciences Seyed Ehsan Enderami Mazandaran University of Medical Sciences Shahram Rabbani Tehran University of Medical Sciences Arefeh Jafarian Tehran University of Medical Sciences Abdolamir Allameh (  allameha@modares.ac.ir ) Tarbiat Modares University Faculty of Medical Sciences https://orcid.org/0000-0003-0757-9572


Background
Type1 diabetes involves the destruction of over 75% of pancreatic islet beta-cells mediated by autoimmune reactions, absolute insulin de ciency, and hyperglycemia [1]. The conventional treatment protocols do not de nitively improve diabetes, but they do help to temporarily control blood sugar levels in a variety of mechanisms [2]. Transplantation of isolated islets of Langerhans from cadaveric donors could be a cure for diabetes [3]; however, this therapeutic strategy is limited by the shortage of organ donors, exhaustion of the transplanted cells and long-term side effects of immunosuppressive agents [4].
In recent years attention has been made on identi cation of stem cells and their application as a renewable source of cells to be differentiated into Insulin-producing cells (IPCs) [5,6]. Adipose-derived stem cells (ADSCs) with high multiplication potency and immune-regulatory properties have attracted signi cant attention for the treatment of diabetes mellitus (DM) [7]. The ADSCs are known for their safety and e cacy compared to embryonic stem cells (ESCs) in both humans and animals, without posttransplant malignancy [8].
In general, the success of in vivo transplantation relies on various factors, among which the matrix and material used to transfer and support the cells and their function is the most important factor. In case of IPCs, the main concern for grafting is to preserve the morphology of the cells in form of cell clusters. Therefore it was assumed that the micro uidic system can protect the cell viability, morphology and functions [9].
Alginate hydrogel with calcium ion solution has been widely used as a scaffold for cell encapsulation using ow-focusing micro uidic device [10]. For example, Jun et al., (2013) have successfully showed that islet like clusters (ILCs) encapsulation with alginate was applied to immune protection of transplanted islets [11].
It has been suggested that speci c miRNAs can also help in improvement of IPCs and their use in cell and molecular therapy. Certain miRNAs are known as important regulatory molecules in multiple processes, including the fate of beta-cells, cell proliferation, differentiation, survival, and apoptosis [12].
Also, several miRNAs are known to participate in beta-cells functions such as insulin expression and secretion, glucose metabolism [13].
Recently the miRNAs with positive and negative regulatory activities on the insulin and IPCs function and their implications in diabetes complications such as cardiomyopathy, nephropathy, and neuropathy has been reviewed [6]. It has been reported that, miR-375 is highly expressed in beta-cells and plays an important role in controlling insulin gene expression and secretion and compensatory beta-cells proliferation and differentiation [14]. It has also been reported that miR-375 plays a signi cant role in maintaining beta-cell mass and regular glucose homeostasis by preserving normal beta-cells and betacell mass proportion [15]. It has been reported that by targeting myotrophin (Mtpn), miR-375 can reduce insulin exocytosis and secretion [16]. This molecule is also responsible for repression of 3phosphoinositide-dependent protein kinase-1 (Pdk1) [17] that might affect the downstream insulin signalling [18]. mir-7 is another miRNA that has been involved in the control of endocrine pancreas development [12]. Wang and his group showed that differentiation of beta-cells and pancreas development is affected by mir-7 through targeting paired box6 (Pax6) and interference inactivation of mTOR pathways [19]. It has also been reported that the miR-7 can inhibit insulin granule secretion by targeting Myrip (myosin VIIA and Rab interacting protein) [20] as a partner of small GTPase Rab27 and Pax6 is a transcriptional factor regulating insulin biosynthesis and secretion [21].
Based on these reports, it appears that some miRNAs is considered as a useful approach for differentiation of IPCs [22]. The present study was designed to gure study the impact of up-regulation of miR-375 and down-regulation of miR-7 during the differentiation of IPCs from the ADSCs and their response to different glucose concentrations. The experiment was pursued by encapsulation of the IPCs using Collagen-Alginate micro ber which mimics the native microenvironment of islets in the pancreas.
The micro bers were then transplanted to a mouse model of diabetes, to see if the micro uidic system can help to improve the functional beta-cells as a therapeutic option for diabetic condition. Adipose tissue was obtained from the abdominal cavity of Balb/c mice and digested for 45min at 37°C in PBS, pH=7.2 (Gibco, Germany), containing 2% BSA (Sigma-Aldrich, USA), and 0.2% collagenase type-І (Gibco, Germany). The isolated ADSCs at a density of 2×10 5 cells/cm 2 were seeded into T 25 culture asks and incubated at 37°C, 5%CO 2 .
The ADSCs at passage three with appropriate monoclonal antibodies labelled with FITC or PE (BD Biosciences, USA) were analysed using uorescence-activated cell sorting on a FACS Caliber (Becton-Dickinson, FAC scan, San Jose, CA, USA).
To con rm the adipogenic and osteoblastic differentiation potential of ADSCs, the cells were subjected to differentiation process and then, the cell were stained with Oil-Red-O and alizarin red (Sigma-Aldrich, USA) respectively and examined under phase-contrast microscopy (Olympus, Japan). The cells were also examined during osteogenesis for alkaline phosphatase activity using BCIP/NBT reagent (Becton Dickinson, Bioscience, UK) for 10-15 min [23].
HEK-293T cells (purchased from Stem Cell Technology Research Center, Tehran, Iran) were seeded in 6 cm Petri dishes with DMEM+10% FBS. The lentivirus carrying miR-375 and anti-miR-7 were propagated from the co-transfection in the HEK-293T cell line using the lipofectamin 2000 transfection reagent (Invitrogen, USA). The titer of the concentrated viral particles was determined using owcytometry. To determine the titer of the GFP-expressing virus, a serial dilution was prepared and added to HEK-293T cells in culture. The transducing unit (TU/ml) of the GFP-expressing cells was determined for each dilution after 48 hours. A well containing HEK-293T cell without the viral dilution was also taken and considered as the negative control.
The multiplicity of infection (MOI) of 30 was considered appropriate for all the experiments. The cells were cultured for one week in fresh complete serum-free medium containing DMEM supplemented with BSA (15%) and 2μg/ml puromycin (Sigma-Aldrich, USA).

Expression of miR-375, miR-7, and its gene targets in the transduced cells
The transduction e ciency was examined by GFP expression in the transduced cells under a uorescence microscope. For determined the miR-375, miR-7 expression, total RNA was extracted from the cells using Trizol reagent (Invitrogen, USA) as instructed by the manufacturer, on day 4 of transduction. In following the experiment, total cellular RNA was isolated from transduced and control cells on days 7, 14, and 21 post-infection using Trizol for determined the pancreatic speci c genes. The quality and quantity of the extracted RNA samples was checked on a Nanodrop (Thermo sher, USA). cDNA was synthetized from each RNA sample (2ng) using a cDNA synthesis kit according to the instructions given by the company (iNtRON Biotechnology, Korea). The expression of genes was determined using quantitative real-time PCR (QRT-PCR) using SYBR green kit (Takara, Korea). The assay was performed in triplicate in a reaction mixture on a real time PCR system (Applied Biosystems, ABI-7500). PCR reaction was performed with mmu-miR-375, mmu-miR-7 primers, and U6 small nuclear RNA endogenous control primers. The qRT-PCR cycling condition was as follows: 95 C for 10 min, followed by 40 cycles (each cycle for 15 sec at 95 C and 1 min at 60 C). The primers used in this experiment are as listed in Additional le 1: Table S1. The relative quanti cation (ΔΔCt) method was applied to calculate the data. Using this assay the gene transcripts of IPCs were compared with that of a mouse pancreatic betacell line (MIN-6, purchased from Iranian Biological Resource Center, Tehran, Iran).

Visualization of the spheroid IPCs and immuno uorescence staining
Formation of the spheroid IPCs obtained on day 21 of differentiation from ADSCs was evaluated by dithizone (DTZ) staining [24]. The cells were further characterized by showing speci c protein localization by ICC technique [25]. The IPCs were treated with primary antibodies against Insulin (#ab7760, Abcam, Cambridge, MA, UK), Glucagon (#ab10988, Abcam), Pdx1 (#ab84987, Abcam), and Neurogenin3 (#ab87108, Abcam). The FITC-coupled goat anti-mouse IgG (#AF8032, Razi Biotech, Iran) and Texas Red-labelled goat anti-mouse IgG (#ab175473) were used as secondary antibody. Besides, samples were processed without primary antibody and considered as negative control. All the ICC assays were performed in triplicate. Wherever indicated, the nuclei of the cells were stained with DAPI (0.1μg/ml).

Estimation of insulin and C-peptide secretion by IPCs
Insulin and C-peptide levels secreted by the IPCs were measured using an ultrasensitive mouse ELISA kit (#Mecodia, Uppsala, Sweden, and # Alpco, Salem, USA respectively). The assays were carried out according to the procedure described by the manufacturer's instruction. Glucose-stimulated insulin release was assayed in differentiated cells after the cells were incubated for 2 hours in freshly prepared Krebs-Ringer bicarbonate buffer (KRB) (Sigma-Aldrich, USA) without glucose. Then the cells were incubated for 2 hours in KRB containing 0.5mmol IBMX (Iisobutyl-3-methylxanthine (Sigma-Aldrich, USA) and different glucose concentrations (5, 10, 15, 20, 25, and 30 mmol). In case of insulin and C-peptide assays in the cells, the cells were rst washed for three times with PBS, extracted in 0.2ml acid alcohol (10% glacial acetic acid in absolute ethanol) at 4°C overnight, and sonicated brie y before centrifugation at 3500 g for 15 min at 4°C. Total protein concentration was determined by BCA protein assay system and 50μg of protein was used for detection of intracellular insulin and C-peptide in each well of ELISA kit.
Absorption was recorded at 450nm and all the assays were carried out in triplicate.

Assembly of micro uidic device and Collagen-Alginate micro-ber
In this experiment, micro uidic device was fabricated using standard soft lithography methods [26,27].
Cylindrical and coaxial-ow channels were prepared by aligning and bonding Poly dimethyl siloxane (PDMS) channels by oxygen plasma treatment (Harrick Scienti c, Ossining, NY) to produce Collagen-Alginate bers. The diameters of the cylindrical channel at the inlet, tapered junction, and outlet were 400, 200, and 600 μm, respectively. In this device, solutions were supplied from the inlets: a sample uid (cells suspended in Collagen-Alginate solution), and a sheath uid (3% (w/v) calcium chloride, dissolved in deionized water). Sodium alginate (Sigma-Aldrich) was prepared in culture medium and collagen by mixing collagen solution and alginate and gently pipetting with cell pellets at a concentration of 1.5×10 6 cells/ml. The ow rate of each uid was adjusted to 100μl/min with the help of a syringe pump and the sheath ow rate was about 1ml/min. The extruded bers were collected in a Petri dish containing CaCl 2 and incubated in culture medium at 37˚C for 45min.

Characterization of the micro ber
Live/dead staining: The viability of encapsulated IPCs was evaluated at 1 and 7 days after encapsulation using mixture of acridine orange (100 μg mL −1 ) and ethidium bromide (100 μg mL −1 ) and viewed using a uorescence microscope. Acridine orange penetrates into cells and stains the DNA of live cells green uorescence using blue lter, whereas ethidium bromide only enters dead cell membranes, stains the DNA of dead cells using green lter and generates a red uorescence. Two images taken from the same eld were merged by Photoshop8 CS software.
Cytotoxicity assessment: Cytotoxicity of the Collagen-Alginate was assessed using 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The differentiated encapsulated cells were soaked in DMEM supplemented with 5% FBS. The cell proliferation was evaluated during 14 days in Collagen-Alginate ber. Brie y, culture medium was removed and replaced with 20μl MTT (0.5mg/ml) (Sigma) was added to each well and plates were incubated at 37°c in dark. The assay was carried out in triplicate, and the results are presented as mean ±SD.
Microstructural of ber (SEM): The surface of the freeze-dried bers was examined using SEM (Philips XL30, Amsterdam, Netherlands). The porosity of bers was measured using ImageJ software.
Fourier Transform Infrared Spectroscopy (FT-IR): The chemical structure of the synthesized hydrogels was investigated by using FT-IR spectroscopy (FT-IR 8400S, Shimadzu, Japan) in the wavenumber of 400-4000 cm −1 .
Swelling property was examined by doing the cross-linked composite porous bers.
The bers were weighed every 50 min and then immersed in excess of swelling medium (pH=7.4) at 37•C until they reach the equilibriums. At various time intervals, the hydrogel was removed from the solution and weighed after blotting the excess water using blotting paper. The weight of the swollen bers was recorded every 50 min until they reach equilibrium state. Data presented in this paper are mean values of triplicate measurements. The percentage swelling of bers was calculated according to the following equation: Q = (Ms -Md) / Md. Where, Q is the swelling ratio, Ms is the mass in the swollen state and Md is the mass in the dried state.
Wherever indicated Type1 diabetes was induced in mice by administration of streptozotocin (STZ) treatments. Each mice received intra peritoneal (I.P) four consecutive injections of STZ (80mg/Kg dissolved in citrate buffer, pH=4.5). Blood glucose level (non-fast) was readily monitored using a portable glucometer on blood samples collected from the tail vein (Accu-CHEK, Roche). Blood glucose was measured at different time intervals and considered as an index for hyperglycemia (diabetes). Blood glucose above 17mmol/l was considered as abnormal which was achieved 5 days after the STZ administration. The animals were selected for transplantation received approximately 1.5×10 6 differentiated and non-differentiated cells encapsulated by the Collagen-Alginate bers. Each mouse received subcutaneous implantation of ber-entrapped cells on the back in the anaesthetized status using 50 mg/kg ketamine and 5 mg/kg xylazine.
The functional e cacy of the transplant was evaluated after four weeks as shown by glucose tolerance test (GTT). Change in blood glucose in fasting mice was monitored for 6-10 hours at different time intervals (20,40,60, 90 and 120 min) following treatment with 2g glucose/kg body weight injected I.P. After 5 weeks of post transplantation (at the end of the treatment period before sacri cing), animals were anesthetized with an I.P. injection of ketamine (70 mg/kg) and xylazine (10 mg/kg) and 2ml of blood sample was collected by heart puncher and used to measure insulin level.

Statistical analysis
The statistical analysis was performed using One-way ANOVA and Bonferroni's post hoc test by Graph pad Prism5 software (GraphPad Software Inc., La Jolla, CA). The results are presented as mean ±SD and P-value less than 0.05 was considered statistically signi cant.

Characterization of the ADSCs
The results of owcytometry analysis of surface markers of the ADSCs isolated from the adipose tissues showed that the cells are positive for CD73 (92.1%), CD90 (90.7%), CD44 (89.3%) and CD105 (83.6%); Whereas the cells were negative for CD45 (0.14%) and CD34 (0.43%) markers. The ADSCs were further characterized after they were induced for osteogenic and adipogenic differentiation of ADSCs. Following differentiation, on day 21, the osteoblasts were identi ed by their calcium deposits using Alizarin Red S stain. Accordingly, alkaline phosphatase activity was detected in the cells using BCIP/NBT (Becton Dickinson, Bioscience) as a substrate. In the case of adipogenic differentiation, the cells obtained on day 14, appeared with oil droplets stained with Oil-Red-O (Additional le 2: Fig S1).

The impact of miRNAs transduction on ADSCs differentiation into IPCs
ADSCs were transduced with lentiviral vectors with an e ciency of about 75% as determined by owcytometry analysis and uorescent microscopy ( Fig.1 a&b).
The expression of miR-375 was sharply increased (70-80 folds; P<0.05) in ADSCs following transduction with a lentiviral vector carrying this gene as compared to cells transfected with miRNA backbone (ADSCs null as control). The expression of miR-7 in ADSCs received anti-miR-7 was dropped by 4-5 folds (P<0.05), as compared to ADSCs null and ADSCs control (Fig.1c&d).
The expression of Mtpn as the target of miR-375 was found to be inhibited in cells received miR-375 or miR-375 along with anti-miR-7. During the three weeks experiment, a noticeable decrease was observed in the gene expression after 7 days. The gene expression was further decreased by 30% (P<0.001) in cells collected on day 14, as compared to the control group. In contrast, expression of Myrip speci c mRNA as a miR-7 target was substantially increased (~40 folds) in cells treated with anti-miR-7 or the anti-miR-7+miR-375 (Fig.1e&f).
Following transduction of miR-375 and anti-miR-7 lentiviruses into the ADSCs, after four days of infection, the adherent spindle-shaped cells started to form round-shape structures by assembling together. Such structures were gradually enlarged during two weeks of transduction in ADSCs carrying miR-375 or miR-375+anti-miR-7. Ultimately the larger structures formed were converted into cluster of IPCs; whereas, under similar conditions, stem cells carrying anti-miR-7 failed to form such structures. On day 21, the ADSCs expressing miR-375 and miR-375+anti-miR-7 were positively stained with DTZ as the indicator of the accumulation of zinc ions present in insulin molecules was stained red. The cells carrying anti-miR-7 alone were negatively stained with the DTZ (Fig.2).

Expression of pancreatic-speci c genes in transduced cells
Time-course studies showed that the expression of the pancreatic transcription factors, namely, Pdx1, Neurog3 were signi cantly increased in ADSCs, received miR-375 or miR-375+ anti-miR-7. Likewise, there was a signi cant induction in expression of endocrine-related marker genes, including Ins1, Ins2, Gcg, and beta-cells genes like Glut 2. (Fig.3). The expression of all the genes was observed seven days after the transduction, and the expression reached to maximum levels on day 14 after induction, after which the levels were gradually declined in ADSCs received miR-375 or miR-375+anti-miR-7. In contrast, the expression of the above-mentioned genes was unaffected in cells transduced with anti-miR-7 assuming that the miR-7 was blocked.

Localization of pancreatic-speci c markers in IPCs (ICC data)
Comparison of immune-staining scoring of Insulin, Pdx1, Ngn3, and glucagon in the cells transduced with miR-375 and miR-375+anti-miR-7 on day 21 of transduction, showed that the accumulation of the markers was relatively lower in cells transduced with miR-375 compared to miR-375+anti-miR-7 (Fig.4a).

Assessment of functional activities of differentiated IPCs
In this experiment, the cells bearing miR-375 lentivirus did not show response to various glucose concentrations (5 to 30mmol) in terms of secretary insulin and C-peptide levels. However, the levels of intracellular insulin and C-peptide were signi cantly elevated in insulin-producing cells that carry miR-375. Also, the levels of secretary and intracellular insulin and C-peptide were signi cantly elevated in differentiated IPCs mediated by miR-375 over-expression and then miR-7 down-regulation (ADSCs miR-375+anti-miR-7) group by different concentrations of glucose from 5 to 30mmol (Fig.4b, c, d &e).

Characterization of IPCs encapsulated with micro bers
The micro uidic chip and its round micro-channels with dimensions are as shown in gure 5. The produced bers were approximately 200μm in diameter, build based on the cluster size of IPCs which were approximately 80 to 180 μm in size. Higher magni cation images taken by SEM revealed a rough and porous surface on the ber preparation and the bers porosity was calculated about 44.8%.
The FTIR spectra of the bers showed two typical peaks of Collagen-Alginate, asymmetric tensile vibration at 1637 cm-1 and another symmetric tensile vibration at 1412cm-1. Both the characteristic peaks move to 1600 cm-1 and 1427 cm-1, respectively, which is related to the cross-linking of carboxyl groups (COO) with the CaCl 2 agent. On the other hand, absence of a clear peak in the range of 2800 cm-1 to 3000cm-1, related to -CH vibration could be assigned to the proper interaction of alginate with collagen and the uniform alloy formation of the two.
The swelling behaviour data for the ber cell-free constructs are as shown in Fig.5i, showed a rapid swelling of the bers which occurred during the early incubation of 100 min, which after 150 min; the swelling tendency was stable for all the samples.
The uorescence images were taken seven days after in vitro cultivation by using a live/dead assay reagent. It was found that alginate-collagen ber entrapped cells has signi cantly more number of cells count on day seven compare to on day one thereby the collagen-alginate does not show inhibiting effect on cells proliferation. The viability of the ber-entrapped IPCs was signi cantly increased as compared to that estimated in ber-free IPCs (control group) on day three, which continued steadily for up to 14 days, indicating the support of the bers for cell growth promotion and proliferation (Fig.6).

The impact of the ber-encapsulated IPCs transplantation on diabetic mice
Blood glucose level in untreated (normal) mice was 4.213±0.33mmol/l, which was sharply increased to 23.9±0.45mmol/l (5-fold) in the diabetic group. After the characterization of the ber, the bers entrapped IPCs were transplanted intra-dermally to 5 diabetic mice.
After four weeks, there was a signi cant decrease (mean=11.027±0.88mmol/l, p<0.0001) in blood glucose when compared to the control group of mice, which were transplanted with ber entrapped undifferentiated ADSCs (22.9±0.97mmol/l, p<0.0001). Moreover, four weeks after transplantation, the GTT data showed that the pattern of blood glucose levels in the mice receiving ber entrapped IPCs was similar to that of non-diabetic mice and increased fasting blood glucose (FBG) which was initially raised, was lowered to 13.8±2.2 and 6.47±0.39 (p<0.0001) in mice treated with ber entrapped IPCs and nondiabetic group respectively. Under this condition, insulin level was signi cantly elevated (266.7±39.11pg/ml), 5 weeks after transplantation to mice as compared to diabetic group with blood insulin level of 162±19.5pg/ml (Fig.7).

Discussion
In the present study, a micro uidic system was designed and applied to improve the survival rate of the functional IPCs differentiated from ADSCs. For this purpose, the ADSCs were used as progenitor stem cells knowing that these cells are an excellent source for the generation of IPCs, which represent a promising cell-based therapy in regenerative medicine and autoimmune diseases [28]. During the differentiation process the cells were forti ed with regulatory miRNAs which presumably target insulinrelated genes. The study was continued with encapsulation of the IPCs using engineered micro ber micro uidic applicable for transplantation and grafting to the host. The data presented here show that during the ADSC differentiation, there were morphological changes together with the increased levels of insulin. The differentiation-dependent changed in morphological features of the ADSCs transduced with miR-375 and anti-miR-7, was more apparent. Signi cant morphological changes observed after four days of miR-375 and anti-miR-7 transduction were due to the conversion of spindle-shaped adherent stem cells into round-shape structures after gathering into clusters of IPCs. This data further support the in uence of miR-375 in proliferation, differentiation, and regulation of insulin expression in beta-cells. In this line, evidences suggest that a moderate reduction (25%) in beta cell mass is unlikely to cause insulin de ciency and diabetes [14,15,29]. Moreover, formation of the IPCs during the differentiation process was con rmed by showing the over-expression of pancreatic-speci c genes, namely, Pdx1, Neurog3, Ins1, Ins2, Gcg, and Glut 2 . This was further con rmed by showing the overexpression of target genes in the IPCs transduced with miR-375 or miR-375+anti-miR-7. The expression of the genes was noticed seven days after the transduction which reached maximum levels on day 14 after induction. A decline in the expression of insulin-related genes occurred after 14 days, which is probably due to a decline in the cell population. The impact of the up-regulation of miR-375 and down-regulation of miR-7 in ADSCs was indicated by ICC. This was further supported by showing that nuclear localization of Pdx1, Neurog3, and cytoplasmic localization of Insulin and Glucagon in differentiated IPCs on day 21 in ADSCs miR-375+anti-miR7 groups. This data implies that targeting certain pancreatic-speci c genes by miR-375, the differentiation process of the ADSCs into IPCs in the absence of cytokines and stimulatory growth factors can be increased. Several transcription factors such as Pdx1, Neurog3, Sox9, and Pax6 are known to control pancreas development and play important role in beta-cell mass, likewise, miR-375 and miR-7 by targeting Pdx1 and Pax6 genes are important in pancreatic development and beta cell function [25,30,31].
As a consequence of transduction of miR-375 and, or anti-miR-7 via lentivirus infection, there was a signi cant increase in the rate of insulin accumulation in the cells and insulin secretion into the culture media. Under such circumstances by augmentation of glucose concentration in IPCs, the levels of secretary and intracellular insulin and C-peptide were also signi cantly increased. The data in Fig.1 clearly attest to the contribution of miR-375 over-expression and then miR-7 down-regulation in regulation of ADSC differentiation. Nevertheless, accumulation of intracellular insulin in IPCs with little secretion in cells transfected with miR-375 lentivirus alone could link to decreased Mtpn expression.
Based on the in vitro assays, it appears that the up-regulation of miR-375 plays an important role in the aggregation of IPCs cluster and the regulation of insulin synthesis by these cells during their differentiation from ADSCs. As a consequence of overexpression of miR-375 in the IPCs, and signi cant decrease in Mtpn expression (the protein target of miR-375 which involves in vesicle exocytosis from beta-cells), could be responsible for impairment in Glucose-stimulated insulin secretion (GSIS) in ADSCs.
The insulin secretion is believed to be related to beta-cell maturation. Also, overexpression of miR-375 by targeting Mtpn could result in decreased insulin exocytosis and secretion [17].
Evidences also show that inhibition of miR-7 in differentiated IPCs is linked to Pax6 mediated increase in GSIS and insulin transcription genes known as genes 1 and 2. Also, a signi cant increase in Myrip expression, as a consequence of mir-7 suppression, may suggest that increased Myrip is can lead to downstream changes, such as the formation tripartite complex with Rab27a and myosin and insulin granule transportation and secretion [32,33].
The use of ber for graft transplantation in diabetic mice is implicated in the improvement of insulin release and improvement of glucose levels in vivo. The advantages of using encapsulation material using collagen-alginate composite (CAC) for uniform-size islet spheroids for in vivo transplantation to diabetic animals has been elucidated. The application of CAC using micro uidic device for islet transplantation with emphasize on immune-protection of the transplanted islet further support the use of CAC [11].
Based on these ndings, the Collagen-Alginate packed matured IPCs given subcutaneously to STZ induced diabetic mice could substantially modulate blood glucose level by lowering its level to 11.02±0.88mmol/l after four weeks, which was abnormally elevated in diabetic mice. In contrast, there was no sign of blood glucose depletion in mice transplanted with undifferentiated ADSCs alone (control).
The glucose lowering ability of the IPC transplantation using collagen-alginate system was associated with increased level of blood insulin in diabetic mice when compared to the control groups. Among the factors contributing to the e ciency of in vivo transplantation was the number of cells transplanted. It is assumed that the adequate number of cells and IPCs which can enhance the e ciency of transplantation is approximately, 1.5× 10 6 . The number of the cells depends on the rate of the ADSCs conversion to IPCs, which is considered important for the e ciency of the graft [9].
According to Silva et al., encapsulation using alginate hydrogels is a strategy being explored as a potential cellular therapy for type-І diabetes conditions without the need for immunosuppression.
Encapsulated allogeneic/xenogeneic transplantation of islets shows promise in the in vivo experiment with blood glucose levels being normalized for extended periods [34]. Perhaps, the encapsulation of the cells using the composite of alginate and collagen mimics the micro-invertebrates of functional beta-cells in the pancreas. The alginate hydrogel membrane could facilitate insulin secretion; in addition, bers by preventing the penetration of immune cells can temporarily protect the cells from cellular immunological challenges. This procedure could facilitate exocrine secretion of insulin as well as allow the penetration of oxygen, nutrients, and glucose inside the micro ber [11,35]. Therefore, the collagen content of the micro ber is also considered as an important factor in supporting the survival and optimal functions of the IPCs. This nding is also supported by studies showing that macro-encapsulation of pig islet cells supports the graft survival and function by increasing oxygenation and neo-angiogenesis [36,37].
In this experiment, the swelling ratio and retention properties are critical indicators of body uids and for the transfer of cell nutrients and metabolites inside the scaffold. These properties have con rmed the stabilization of the shape and size of the scaffolds during in vitro cell culture and in vivo implantation [38,39].The advantages of using encapsulation material using collagen-alginate composite for uniformsize islet spheroids for in vivo transplantation to diabetic animals has been elucidated [40].

Conclusion
In conclusion, the results presented here clearly show that miR-375 and, or anti-miR-7 play important roles in differentiation of ADSCs into mature and functional IPCs. The IPCs responsive to glucose challenge did not elicited following the up-regulation of miR-375 alone, whereas, suppression of miR-7 following over expression of miR-375 caused signi cant changes in glucose response and insulin secretion. In vivo experiment showed that the use of advanced cell therapy systems with Collagen-Alginate ber-entrapped IPCs is associated with supporting the IPC survival and better functions to control hyperglycaemic condition, suggesting that the encapsulation of IPCs is promising for successful allograft transplantation. Expression of miR-375 and miR-7 in test and control groups was measured by qRT-PCR on day 4 after transduction (e& f) Detection of the expression level of target mRNAs, Mtpn, and Myrip, were analyzed at different days of differentiation into IPCs in test and control groups by qRT-PCR. All tests were performed in triplicate, and data were presented as mean ±SD. * P <0.05. like clusters began to appear, and after 14 days, the number of matured aggregates increased, and at the end of day 21, some clusters were detached from the plate and died (scale bars are 100μm). At this time, differentiated cells that were positive for DTZ staining appeared (scale bars are 50μm).

Figure 3
Expression of pancreatic endocrine genes in insulin producing cells derived from adipose-derived stem cells (QPCR). The expression levels of pancreatic transcription factors such as Pdx1 and Neurog3, endocrine markers: Ins1, Ins2, Gcg, and beta-cells speci c gene like Glut2 were analysed at each stage of differentiation into IPCs. Gene transcripts of IPCs were compared with undifferentiated ADSCs null (negative control) and MIN-6 cell line (positive control). Relative levels of gene expression were normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript value is shown in each graph as mean ±SD. * P <0.05.