Two rats were used for stem cell isolation, culture, phenotyping and labeling. In addition, a part of pancreas was homogenized for SERCA2b preparation. Twenty rats were used for isolation of the islets from the rats’ pancreases to assess the islet calcium level. They were subdivided into 4 subgroups (5 rats each).

It included 6 rats that corresponded to and were sacrificed with the experimental groups II, III and IV respectively. The first 2 rats received a single intraperitoneal (IP) injection of 0.5 ml citrate buffer. The second 2 rats received a single IP injection of 0.5 ml citrate buffer. The day following confirmation of diabetes, 1 ml of phosphate buffered saline (PBS) was given intravenous (IV) via tail vein. The third 2 rats received citrate buffer and PBS as for the 2^nd 2 rats, in addition to 1.5 μl lipofectamine via the tail vein.

It included 10 rats. Diabetes was induced by a single IP injection of STZ (Sigma Company, St. Louis, Mo, USA) in a powder form as 1 g vial. The required dose (50 mg/kg body weight) (8) was weighed using a digital scale and dissolved in 0.5 citrate buffer for each rat. Three days following STZ injection, diabetes was confirmed by measuring the blood glucose level. The animals were considered diabetic if their blood glucose level was higher than 200 mg/dl (9).

It included 10 rats. On the day following confirmation of diabetes, 1×10⁶ of cultured and labeled rat AMSCs, suspended in 1 ml PBS, were injected into the tail vein (10).

It included 10 rats. On the day following confirmation of diabetes, 1×10⁶ of cultured and labeled SERCA2b transfected AMSCs were injected as in group III.

In group I (control) and the corresponding experimental groups II, III and IV, the rats were sacrified 3 weeks following the day of confirmation of diabetes.

Under sterile conditions, rats were euthanized with carbon dioxide (CO₂), their abdomens were cut open and the adipose tissues were removed. After the adipose tissues were washed with saline solution, all the tissues were collected and incubated in Dulbecco’s modified Eagle’s medium (DMEM), (GIBCO/BRL) supplemented with 10% fetal bovine serum (FBS) (GIBCO/BRL). Nucleated cells were isolated with a density gradient [Ficoll/Paque (Pharmacia)] and resuspended in complete culture medium supplemented with 1% penicillin-streptomycin (GIBCO/BRL). Cells were incubated at 37°C in 5% humidified CO2 for 12~14 days as primary culture or upon formation of large colonies. When large colonies developed (80~90% confluence), cultures were washed twice with PBS and cells were trypsinized with 0.25% trypsin in 1millimolar EDTA (GIBCO/BRL) for 5 minutes at 37°C. After centrifugation (at 2400 rpm for 20 minutes), cells were resuspended with serum-supplemented medium and incubated in 50 cm2 culture flask Falcon). The resulting cultures were referred to as first-passage cultures. On day 14, the adherent colonies of cells were trypsinized, and counted.

Adherent cells (at the end of 3rd passage) were trypsinized and adjusted to 1×10⁶ cells/ml determined by hemocytometer and incubated with 10 μl of monoclonal antibodies: CD34 and CD44 (Beckman coulter, USA) at 4°C in the dark. After 20 minutes incubation, 2 ml of PBS containing 2% fraction crystallizable solution (FCS) were added to each tube of monoclonal treated cells. The mixtures were then centrifuged for 5 minutes at 2500 rpm followed by discarding the supernatant and resuspending cells in 500 μl PBS containing 2% FCS. Cell analysis was performed using CYTOMICS FC 500 Flow Cytometer (Beckman coulter, FL, USA) and analyzed using CXP Software version 2.2 (10). Immunohistochemical characterization was performed by using streptavidin immunoperoxidase technique for CD44 and CD34 (13).

Non-transfected AMSCs cells were harvested during the 4th passage and were labeled with PKH26 fluorescent linker dye to be examined by fluorescent microscope.

Pancreatic tissues were homogenized in a solution containing (mM) 250 sucrose, 5 hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES) and 0.2 phenylmethanesulfonyl fluoride (PMSF), using a Polytron homogenizer. Ca²⁺⁻dependent Ca²⁺⁻ATPase activity in homogenates was measured at 37°C. The data were analyzed by nonlinear regression with computer software (GraphPad Software). Processing for ribonucleic acid (RNA) extraction was followed by using reverse transcriptase (for complementary DNA (cDNA) synthesis (15).

The cells were plated at a confluency of 50% (500,000 cells/plate) one day prior to transfection. 1.5 μl Lipofectamine 3000 reagent (Invitrogen) and 1.5 μg SERCA2b labeled with green fluorescent protein (GFP) were used for transfection using serum-free DMEM. GFP directly fuses to selected locations on SERCA2b (17). Culture media were changed 4 hours after transfection and replaced with DMEM/10% FBS without antibiotics. To assess transfection efficiency, cells labeled with GFP are first visualized via fluorescence microscopy for qualitative assessment of protein expression, morphology and viability. Cells were then prepared for flow cytometry by aspirating the medium and replacing it with 250 μl of a 7 : 3 mixture of TrypLE reagent: 1 X DPBS. Cells were incubated at 37°C for 10 min and then pipetted up and down to ensure single cells for flow cytometry analysis. The cells were then allowed to proliferate and differentiate in DMEM/2% FBS.

Pancreatic islets were aseptically isolated from rat pancreases according to the optimized protocol described by Shewade et al. (16). Aseptically excised rat pancreases were minced into three 1-mm pieces and digested with collagenase (Sigma Aldrich, USA) (1 mg/ml) for 10 min. The collagenase was then inactivated with two washes of RPMI 1640 containing 10% fetal calf serum (Sigma Aldrich, USA) and the samples were seeded into the same medium at one pancreas per flask. The primary cultures were incubated at 37°C under 5% CO2 for 48 h. The isolated islets of the 1st subgroup were considered control. The isolated islets of the 2nd subgroup were exposed to STZ (5 mM) for 1 h at 37°C. The STZ solution was prepared in phosphate-buffered saline (Sigma Aldrich, USA). The isolated islets of the 3rd subgroup were exposed to STZ (5 mM) for 1 h and then treated with AMSCs (10 μM). The isolated islets of the 4th subgroup were exposed to STZ (5 mM) for 1 h and then treated with SERCA 2b transfected AMSCs (10 μM).

The calcium level was assessed in the islet culture medium after treatment by colorimetric methods according to the kit instructions. The analytical kits were supplied by Quimica Clinica Aplicada SA (Amposta, Spain).

Before sacrifice tail vein blood samples were collected at the end of the 3^rd week from groups I, II, III and IV for blood glucose and plasma insulin levels estimation.

Plasma insulin level was measured by enzyme immunoassay of 25-l aliquots of plasma with a Rat Insulin ELISA kit (DRG, USA). During incubation, insulin in the sample reacted with peroxidase-conjugated anti-insulin antibodies, which were bound to the plastic surface of the microtitration well. The bound conjugate was detected by reaction with 3, 3′, 5, 5′-tetramethylbenzidine. The reaction was stopped by adding acid to give a colorimetric end point that was read spectrophotometrically (20).

The animals were sacrificed by cervical dislocation (21). A central ventral midline incision was performed for each animal. The pancreas specimens were excised and fixed in 10% formol saline for 48 hours. Paraffin blocks and 5 μm thick sections were prepared. For semithin sections, pancreas specimens (1 mm) were processed for embedding in epoxy resin, cut at1 μm thickness and staind with toluidine blue. Sections were subjected to

0.1 ml prediluted primary rabbit polyclonal CD44 antibody was applied to sections for 60 minutes, tonsil sections were considered +ve control and the reaction is membranous. 0.1 ml diluted 1ry antibody rabbit monoclonal insulin antibody was applied to sections for 60 minutes pancreas sections were considered +ve control and the reaction is cytoplasmic,

Using Leica Qwin 500 LTD (Cambridge, UK) computer assisted image analysis system, assessment of the area percent (%) of collagen fibers performed in Masson’s trichrome stained sections. The measurements were done in 10 low power fields (PFs) in control and experimental groups. Insulin IE and CD44 + ve cells were measured using binary mode. The measurements were done in 10 high PFs in control and experimental groups. The areas of chosen colors were masked by a blue binary color inside a standard measuring frame of area.

Evaluation of formalin-fixed paraffin-embedded (FFPE) pancreas specimens by real time qPCR was performed (26). Reverse Transcription is carried out with the SuperScript First-Strand Synthesis System for reverse transcriptase (RT)-PCR. The following procedure is based on Invitrogen’s protocol. The SERCA2b primer using the 5′-oligonucleotide primer 5′-AC GTC AAT GGG TCG ACT ATT TAC G-3′ and 3′-oligonucleotide primer 5′-AGA AGG CAC AGT CGA GGC TG-3′ (mixture was prepared in each tube: 5 μg total RNA and 3 μl random hexamers). The samples were incubated at 65°C for 5 min and then on ice for at least 1 min. Reaction master mixture was prepared for each reaction then added to the RNA/primer mixture, mix briefly, and then placed at room temperature for 2 minutes. 1 μl (50 units) of SuperScript II RT was added to each tube, mixed and incubated at 25°C for 10 min. The tubes were incubated at 42°C for 50 min, heat inactivated at 70°C for 15 min, and then chilled on ice. 1 μl RNAase H was added and incubated at 37°C for 20 min. The 1st strand complementary deoxyribonucleic acid (cDNA) was stored at −20°C until use for real-time PCR. The primer concentrations were normalized, gene-specific and reverse primer pair were mixed. Each primer (forward or reverse) concentration in the mixture is 5 pmol/μl. The PCR program was set up on ABI Prism standard deviation score (SDS) 7000. A copy of the setup file was saved and all PCR cycles were deleted (used for later dissociation curve analysis). 50°C 2 min, 1 cycle, 95°C 10 min, 1 cycle, 95°C 15 seconds → 60°C 30 seconds → 72°C 30 seconds, 40 cycles and 72°C 10 min, 1 cycle. A real-time PCR reaction mixture can be either 50 μl or 25 μl. After PCR is finished, the tubes were removed from the machine. The PCR specificity was examined by 3% agarose gel using 5 μl from each reaction. The setup file was saved and the real-time PCR result was analyzed with the SDS 7000 software. β-actin was used as internal control to normalize values with the following primer (forward primer: 5′-CCT TCCTGGGCATGGAGTCCT-3′ & reverse primer: 5′-GGAGCAATGATCTTGATCTTC-3′ ).

Any significant ANOVA was followed by Bonferroni post-hoc test to detect which pairs of groups caused the significant difference. p-values <0.05 were considered statistically significant. Calculations were made on Statistical Package for the Social Sciences (SPSS) version 16.

AMSCs appeared mostly spindle in shape, immunostaining showed +ve membranous brownish reaction for CD44 (arrows) (Fig. 1A) and −ve for CD34 (arrows) (Fig. 1B). Immunophenotyping of AMSCs by flow cytometry showed 96% of the cells +ve for CD44 (Fig. 1C).

Transfected cells appeared mostly as spindle fluroscent labeled cells (Fig. 1D). Immunophenotyping of transfected AMSCs by flow cytometry showed 93.3% of the cells GFP labeled (Fig. 1E).

The mean calcium level values± standard deviation (SD) were (1.41±0.03) in the control group, (0.77±0.02) in the diabetic group, (1.03±0.04) in the AMSCs group and (1.31±0.03) in the SERCA2b transfected AMSCs group. The previous values indicated a significant decrease in the diabetic group compared to the other groups and in the AMSCs group compared to control and transfected AMSCs group.

Blood glucose level values indicated a significant increase in the diabetic group before sacrifice compared to the other groups. The plasma insulin values revealed a significant decrease in the diabetic group compared to the other groups and in the AMSCs group compared to control and SERCA2b transfected AMSCs groups (Table 1).

In toluidine blue stained sections, all control rats demonstrated islet cells exhibiting pale nuclei and secretory granules (Fig. 2A). Sections of group II recruited shrunken islets exhibiting few cells with absent secretory granules (Fig. 2B). Sections of group III showed condensed nuclear chromatin of few islet cells, multiple pale nuclei and secretory granules in most of islet cells (Fig. 2C). Sections of group IV demonstrated condensed nuclear chromatin of few islet cells, pale nuclei and secretory granules in most cells (Fig. 2D).

In Masson’s trichrome stained sections, group I revealed fine collagen fibers and group II recruited increased collagen fibers between the acini and among the islet cells (Fig. 3A, B). Group III demonstrated minimal increase in the collagen content around vessels and fine collagen fibers among the islet cells and group IV showed fine collagen fibers (Fig. 3C, D).

In fluorescent labeled sections, group III showed some PKH26 fluorescent labelled cells (Fig. 4A) and in group IV few GFP fluorescent labelled cells were evident among the acini and islets (Fig. 4B).

In CD44 immunostained sections, group I showed −ve IE (Fig. 5A). Group II demonstrated multiple +ve spindle cells among the islets and pancreatic acini in few fields (Fig. 5B). While group III recruited multiple + ve cells and group IV some +ve cells among the islets and acini in multiple fields (Fig. 5C, D).

In insulin immunostained sections, group I showed +ve IE among most of the islet cells, in group II shrunken islets recruited +ve IE among few cells, in group III +ve IE was seen among multiple islet cells and group IV clarified +ve IE among most of the islet cells (Fig. 6A, B, C, D).

Concerning the mean area % of collagen fibers, a significant increase was found in group II compared to the other groups. The mean area % of CD44 +ve cells revealed a significant increase in group III compared to groups II and IV, in addition in group IV compared to group II.

The mean area % of insulin +ve IE denoted a significant decrease in group II compared to the other groups and in group III compared to groups I and IV.

The mean values of PCR±SD were (1.93±0.30) in group I, (0.14±0.04) in group II, (0.61±0.08) in group III and (1.59±0.11) in group IV. The previous values indicated a significant decrease in the group II compared to the other groups and in group III compared to groups I & IV.