Sprague-Dawley (SD) rats of closed colony were purchased from Animal Center, Nanjing Medical University. All the procedure was accordant with animal experiment guidelines of the university. BM was obtained from the femurs and tibias of 10 male SD rats (200-250 g) under aseptic condition, separated by Ficoll density gradients centrifugation and dispersed into a single cell suspension. BM cells (1 × 10⁶ cells/mL) were cultured in 75 cm² flask with low glucose (5.6 mmol/L) Dulbecco’s modified eagle’s medium (LG-DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA), HEPES (20 mmol/L), L-glutamine (2 mmol/L), penicillin (100 μ/mL) and streptomycin (100 mg/mL) at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. Suspended cells were disposed 24 h later and adherent cells were cultured in 10% FBS LG-DMEM which was changed every 3 d. BM-MSCs gaining 80%-90% confluence were passaged by digestion with 0.25% trypsin and 0.02% EDTA. Following two to three passages, the cells became morphologically homogeneous.

After the third passage, BM-MSCs were released by trypsinization. The cells were incubated with anti-rat phycoerythrin (PE)-labeled CD45 antibody (1:20) and fluorescein isothiocyanate (FITC)-labeled CD90 antibody (1:20) (Caltag, USA) or FITC-labeled CD29 antibody (1:20) (Biolegend, USA) for 20 min, then resuspended in 1% paraformaldehyde/PBS and acquired onto FACSCalibur (BD, USA), the positive rates were assessed by Cellqust software. Isotypematched rat immunoglobulins served as controls for autofluorescence.

At the third passage, BM-MSCs with 80% confluence were induced to differentiate into pancreatic islet cells. Cells were induced with 5% FBS HG-DMEM (25 mmol/L glucose) for 14 d, and added 10 mmol/L nicotinamide (Sigma, USA) for 7 d, and then 10 nmol/L exendin-4 (Sigma) for 7 d.

During differentiation, morphological changes of BM-MSCs were investigated under a converted microscope. BM-MSCs and differentiated cells (D-MSCs) were fixed in 5% glutaraldehyde for 2 h at 4°C, washed in PBS, transferred to 1% osmic acid for 2 h at 4°C, washed in PBS, then dehydrated in acetonic acid and embedded. Ultra thin sections were counterstained using uranyl acetate and lead citrate, then viewed by electron microscope (JEM-1010, Japan).

Total RNA from pre-induced BM-MSCs, D-MSCs and normal rat pancreas tissue was isolated using TRIzol reagent (Gibco) and pretreated with DNase to remove genomic DNA contamination. Transcriptional gene expressions related to pancreatic endocrine development and function were determined by RT-PCR kit (Promega, USA). GAPDH was used as an internal control. PCR cycles were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, annealing temperature (Tab 1) for 30 s, 72°C for 30 s, and final extension at 72°C for 10 min. PCR products were separated by electrophoresis in 1.0% agarose gels and photographed by Kodak digital camera. The name and sequences of the primers, the sizes of PCR products, and annealing temperature for each pair are listed in Table 1. The primers were synthesized by Shanghai BIOASIA Biologic Technology CO. LTD.

Pre-induced BM-MSCs, D-MSCs and RIN-m5F cells were grown in plastic six-well plates on slide coverslips (22 × 22 mm²). Cells were fixed in methanol for 15 min, washed with PBS, incubated with 0.01% Triton-100 and first antibody for 20 min, washed with PBS, and cultured with secondary antibody for 20 min, washed with PBS. Insulin, C-peptide, glucagons (Gcg), somatostatin (SS) and islet amyloid polypeptide (IAPP) expressions were observed under laser confocal microscope (LSM510, Carl Zeiss, Germany). First antibody: guinea pig anti-insulin (1:50) (Zymed, USA), rabbit anti-Gcg (1:50) (Zymed), rabbit anti-SS (1:50) (Zymed), rabbit anti-IAPP (1:50) (Zymed), goat anti-rat C-peptide (1:50) (Linco Research Inc., USA); Secondary antibody: anti-Guinea pig IgG (1:20) FITC conjugated (KPL,USA), anti-rabbit IgG (1:20) FITC/Cy5 conjugated (KPL), anti-goat IgG (1:20) FITC conjugated (KPL).

Pre-induced BM-MSCs and D-MSCs (n = 10) were fixed in methanol for 15 min, washed with PBS, incubated with 0.01% Triton-100 and Guinea pig anti-Insulin (1:50) for 20 min, washed with PBS, and cultured with anti-Guinea pig IgG FITC conjugated (1:20) for 20 min, washed with PBS, then resuspended in 1% paraformaldehyde/PBS solution and acquired onto FACSCalibur. The insulin expression and mean immunofluorescence intensity were assessed by Cellqust software. Isotypematched rat immunoglobulins served as controls for autofluorescence.

Pre-induced BM-MSCs and D-MSCs (10⁶/mL, n = 5) were switched to serum-free LG-DMEM containing 0.5% BSA for 12 h, washed twice with PBS, then stimulated by HG-DMEM for 2 h. The culture medium was collected and frozen at -70°C. Serum-free LG-DMEM containing 0.5% BSA was used as a control for secreted insulin measurement. Insulin release was detected by rat insulin enzyme-linked immunosorbent assay (ELISA kit, Linco) according to the manufacturer’s instructions.

Hyperglycemia was induced in 16 male SD rats of closed colony (Body weight 180-200 g) through intraperitoneal injection of 60 mg/kg of streptozotocin (STZ). Blood glucose levels were determined using Roche ACCU-CHEK glucose meter. Stable hyperglycemia (blood glucose levels ranging between 16.7-33.3 mmol/L) developed in 14 rats one week later. Under general anesthesia, the rats received a transplant of 5 × 10⁶ D-MSCs (n = 7) or pre-induced BM-MSCs (n = 3) or 600 freshly isolated islets (n = 4) via portal vein. Glucose levels were monitored by tapped tail-vein blood under non-fasting condition every two days after transplantation for 28 d.

Liver and pancreas of the recipient at d 12 after D-MSCs transplantation were removed and fixed in 4% formaldehyde and embedded in paraffin. To detect D-MSCs grafts in the liver, all liver lobes were sampled. Sections were stained with anti-insulin (1:50) (DAKO, Denmark), anti-Gcg (1:50) (Zymed), anti-SS (1:50) (Zymed) and anti-IAPP (1:50) (Zymed), respectively. Immunohistochemical analysis was performed using an EnVision^TM + System-HRP (DAB) (DAKO) following the manufacture’s instruction.

Data are presented as mean ± SD. Results were analyzed using one-way ANOVA. Statistical significance was set at P < 0.05.

Pre-induced BM-MSCs were typical of spindle and fibrocyte-like adherent monolayers (Figure 1A) with high CD90 positive rate (96.3% ± 1.3%), CD29 positive rate (93.9% ± 0.8%) and very low CD45 expression (0.3% ± 0.4%) (Figure 1F). When being switched into 5% FBS HG-DMEM, BM-MSCs began to form three dimensional, islet-like clusters (Figure 1B). After induced by nicotinamide and exendin-4, clusters were increased and some half suspended in the culture medium (d = 80-200 μm) (Figure 1C). An electron micrograph of D-MSCs revealed structures typical of a secretory cell, including rough endoplasmic reticulum, Golgi complex, a few large vacuoles and secretory vesicles containing dense granules (Figure 1E). However, few could be found within the cytoplasm of pre-induced BM-MSCs (Figure 1D).

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Figure 1 Morphological changes of BM-MSCs during differentiation. A: The third passage of pre-induced BM-MSCs (× 500); B: BM-MSCs formed islet-like clusters under 5% FBS HG-DMEM culture (× 500); C: Some clusters were half suspended in the culture medium after induced by nicotinamide and exendin-4 (× 500); Electron microscopy: Secretory granules (the black arrow shows) are densely packed within the cytoplasm of D-MSCs (E, × 4000) whereas few is found in pre-induced BM-MSCs (D × 5000); F: Surface markers of BM-MSCs showed that CD90 and CD29 positive rates were more than 93% whereas CD45 expressions were less than 1%.

Transcriptional gene expressions related to pancreatic endocrine development and function were not detected in pre-induced BM-MSCs. However, when BM-MSCs were induced with 5% FBS HG-DMEM for 14 d, insulin (Iand II), Gcg, SS, IAPP, glucagon-like peptide (GLP)-1 receptor (GLP-1R), pancreatic duodenal homeobox-1 (PDX-1), Ngn3, NeuroD1, PAX-6 and GLUT-2 messages were positively expressed. After adding nicotinamide and exendin-4 for another 14 d, D-MSCs showed the expression of the aforementioned genes and GK, Nkx2.2 mRNAs (Figure 2).

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Figure 2 Gene expressions during BM-MSCs differentiation. M DNA marker: 1 Rat pancreas; 2 Pre-induced BM-MSCs; 3 BM-MSCs in 5% FBS HG-DMEM culture; 4 BM-MSCs in 5% FBS HG-DMEM culture with nicotinamide and exendin-4.

To investigate the expressions of pancreatic islet hormones, immunofluorescence analyses were performed for insulin (INS), C-peptide, Gcg, SS and IAPP in D-MSCs. RIN-m5F cells were shown to strongly express INS, C-peptide, Gcg, SS, but negative for IAPP (Figure 3A). Pre-induced BM-MSCs were negative for the above islet hormones (Figure 3B); However, D-MSCs did express these cytoplasmic proteins (Figure 3C); Some D-MSCs co-expressed INS/Gcg, INS/SS, INS/IAPP (Figure 3D). Flow cytometry showed that the insulin positive rate of cells induced by 5% FBS HG-DMEM for 14 d was about 11.2%. When added with nicotinamide and exendin-4, the insulin positive rate was up to around 19.8% and mean fluorescence intensity was increased by 2.6 folds, which was significantly higher than that of pre-induced BM-MSCs (Figure 4, P < 0.05). To determine whether D-MSCs could secret insulin and response to a glucose challenge, insulin release from pre-induced BM-MSCs and D-MSCs was measured by ELISA. Pre-induced BM-MSCs and cells induced with 5% FBS HG-DMEM for 14 d showed little insulin secretion and glucose response. However, D-MSCs could secret a small amount of insulin (roughly 2.0 ng/10⁶ cells) into medium and increase by 1.5 fold in the presence of glucose challenge (Figure 4).

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Figure 3 Immunofluorescence reveals BM-MSCs differentiated into islet-like cells in vitro. Rat RIN-m5F cells displayed insulin (INS), C-peptide (C-P), glucagons (Gcg), somatostatin (SS), but without islet amyloid polypeptide (IAPP) (A, × 400); Pre-induced BM-MSCs were negative for the above islet hormones staining (B, × 400); D-MSCs showed the staining of INS, C-P, Gcg, SS, IAPP (C, × 400); Some D-MSCs co-expressed INS/Gcg, INS/SS, INS/IAPP (D, × 400).

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Figure 4 Insulin content and release in response to glucose stimulation of BM-MSCs in different groups. Insulin positive rate of D-MSCs was up to around 19.8% and mean fluorescence intensity was increased by 2.6 folds, which was significantly higher than that of pre-induced BM-MSCs. D-MSCs secreted a small amount of insulin into medium and the insulin was increased by 1.5 folds under the glucose challenge. ^aP < 0.05 vs pre-induced BM-MSCs; ^cP < 0.05 vs HG; ^bP < 0.01 vs other group. N: nicotinamide; E: exendin-4.

To determine whether D-MSCs possessed the capacity to correct hyperglycemia in diabetic rats, D-MSCs (5 × 10⁶/rat) were transplanted via portal vein into STZ-induced diabetic rats. Pre-induced BM-MSCs (5 × 10⁶/rat) and freshly isolated islets (600/rat) were transplanted as controls. As demonstrated in Figure 5, glucose levels in D-MSCs implanted rats began to decrease at d 6 after transplantation, kept below 15 mmol/L during d 12 to d 16, and then elevated again after d 20. In contrast, glucose levels in islets implanted rats decreased at d 2, kept below 15 mmol/L during d 4 to d 8, and then elevated again after d 10. Glucose levels in the pre-induced BM-MSCs implanted rats remained elevated (P < 0.05). Immunohistochemical analysis revealed that D-MSCs grafts were located in the recipient’s liver in close proximity to the portal vein and expressed insulin, Gcg, SS and IAPP (Figure 6B). Very few native islets could be found in the pancreas of STZ-induced diabetic recipient rats. In the remaining pancreatic islets, only few insulin-expressing cells located in the center, rich Gcg-expressing cells setting at the periphery, a few SS-expressing cells and IAPP-expressing cells scattering in the islets (Figure 6A).

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Figure 5 Glucose change of STZ-induced diabetic rats after D-MSCs transplantation via portal vein. Glucose levels in the D-MSCs implanted rats began to decrease at d 6 following transplantation, kept below 15 mmol/L during d 12 to d 16, and then elevated again after d 20. In contrast, glucose levels in islets implanted rats decreased at d 2, kept below 15mmol/L during d 4 to d 8, and then elevated again after d 10. Glucose levels in the pre-induced BM-MSCs implanted rats remained elevated. ^aP < 0.05, ^bP < 0.01 vs pre-induced BM-MSCs implanted rats.

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Figure 6 Islet hormones expression in the pancreas and liver of STZ-induced diabetic rat after D-MSCs transplantation via portal vein. D-MSCs grafts were located in the liver (B, × 400), expressing insulin (INS), glucagons (Gcg), somatostatin (SS) and islet amyloid polypeptide(IAPP); In the remaining pancreatic islets, a few insulin-expressing cells located in the center, rich Gcg-expressing cells setting at the periphery, a few SS-expressing cells and IAPP-expressing cells scattering in the islets (A, × 400).