Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells

Abstract

The maintenance of viable and functional islets is critical in successful pancreatic islet transplantation from cadaveric sources. During the isolation procedure, islets are exposed to a number of insults including ischemia, oxidative stress and cytokine injury that cause a reduction in the recovered viable islet mass. A novel approach was designed in which streptozotocin (STZ)-damaged rat pancreatic islets (rPIs) were indirectly cocultured with rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to maintain survival of the cultured rPIs. The results indicated that islets cocultured with rBM-MSCs secreted an increased level of insulin after 14 days, whereas non-cocultured islets gradually deteriorated and cell death occurred. The cocultivation of rBM-MSCs with islets and STZ-damaged islets showed the expression of IL6 and transforming growth factor-β1 in the culture medium, besides the expression of the antiapoptotic genes (Mapkapk2, Tnip1 and Bcl3), implying the cytoprotective, anti-inflammatory and antiapoptotic effects of rBM-SCs through paracrine actions.

Figure 1

Morphology of PIs and rBM-MSCs. (a) Free-floating rat islets (arrows). (b) Rat islets stained with DTZ for specificity. (c) During the onset of growth (P₀-13th day), isolated cells from the bone marrow formed single-cell-derived colonies (arrows). In later passages, most of these MSCs exhibited large, flattened or fibroblast-like morphology (P₁-12th day) (d) (Original magnification: a, c × 40; b, d × 200)

Figure 2

Agarose gel electrophoresis of RT-PCR products. Representative panel of RT-PCR analysis of rBM-MSCs. Internal control: glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Negative control: PCR mix without template

Figure 3

Cell viability. For determination of cell viability in normal (a, b) and STZ-induced pancreatic islets cocultured with rBM-MSCs (c, d), PI/FDA staining was performed. The small bright spots in the fluorescent micrographs correspond to the intense red staining of PI-positive (dead) cells. The diffusely stained regions correspond to the FDA-stained (live) cells (Scale bars=50 μm)

Figure 4

Detection of apoptosis. (a) Apoptosis was quantified by FACS analysis after staining with Annexin V and PI at the end of the experiment (day 14) (islets were incubated with trypsin to obtain dispersed cells). Viable cells were Annexin V−/PI− (III), early apoptotic cells were Annexin V+/PI− (IV), late apoptotic cells were Annexin V+/PI+ (II) and necrotic cells were Annexin V−/PI+ (I). Representative examples are shown in b. (c) We also performed PI/FDA staining for determination of cell viability in normal and STZ-induced pancreatic islets cocultured with rBM-MSCs and calculated as described in Methods. STZ induced a significant decrease in the viability rates in islets compared with untreated islets (10%±2.8, 60%±2.81, respectively). Coculture with rBM-MSCs induced a significant increase in viability rates in STZ-induced islets (72.5%±4.01; P<0.001). (mean±S.D., n=3 each, ^**P<0.01, ^***P<0.001)

Figure 5

Insulin release in response to glucose stimulation of islets in different groups. On day 14, normal islets, islets cocultured with rBM-MCSs, STZ-induced islets and STZ-induced islets cocultured with rBM-MSCs secreted insulin into the medium and the insulin was increased under glucose challenge. However, the insulin secretion level of STZ-induced islets was decreased more significantly than in other groups, similar to under H-DMEM conditions. Interestingly, injured islets cocultured with rBM-MSCs released insulin into culture medium under glucose stimulation, similar to normal islets. Data represent the mean±S.E. of values of three independent measurements. Data were analyzed for significant change by using two-way ANOVA and paired t-test (^*P<0.05; ^**P<0.01)

Figure 6

Cytokine secretion evaluated by ELISA. The expressions of TGF-β1 and IL-6 were quantified in various monocultures including islets, STZ-induced islets and rBM-MSCs and cocultures including islets+rBM-MSCs and STZ-induced islets+rBM-MSCs. Data represent the mean±S.E. of values of three independent measurements. Data were analyzed for significant change by using two-way ANOVA and paired t-test (^*P<0.05; ^**P<0.01)