1% Triton X-100 at room temperature for 30 min. After,
cells were washed in PBS thoroughly. Cells were then incubated with 1 μM phalloidin-rhodamine (Biotium, Inc., Hayward, CA, USA) at 4°C overnight to label F-actin. After several washes in PBS, the labeled cells were scanned by LCSM (510 Meta Duo Scan, Carl Zeiss, Oberkochen, Germany) using 545-nm (He-Ne) excitation. Emission was detected above 600 nm. Statistical analysis All data were presented as mean values ± standard deviation taken from ten different cells. The morphologic parameters between the different groups were compared using t test (via SPSS 11). Differences with P values less than 0.05 were considered to be R788 supplier statistically significantly. ABT-888 mw Results Morphology and phenotypes of cultured hADSCs Isolated hADSCs Selleck AR-13324 exhibited a spindle shape, began to appear in culture, and reached 90% confluence
in about 10 to 12 days. The second passage of hADSCs expanded rapidly and developed a uniform morphology that resembled that of fibroblasts. FACS analysis of hADSCs at the third passage showed that these cultured cells were positive for CD13 (98.88%), CD44 (98.9%), CD59 (98.4%), and CD105 (71.24%). In addition, expression of HLA-DR (0.98%) was not detected. Furthermore, hADSCs exhibited low expression of hematopoietic lineage markers CD45 (1.03%) and CD34 (2.88%). Differentiation of IPCs Insulin cannot be used as a differentiating medium, so the insulin that appeared in media after glucose stimulation was synthesized de novo and secreted by IPCs. Figure 1 shows that the expression of insulin gene massively increased. Insulin mRNA expression in IPCs increased 16-fold, from day 0 to day 12 (P < 0.05). To verify whether IPCs could secrete insulin as a result of sensing physiological glucose concentrations as beta cells do, we first detected the quantity of insulin secretion in different glucose concentrations and under different stimulating time frames. ELISA (Table 2) showed that beta cells and IPCs from all four donors secreted insulin after 30 min or 1 h of stimulation, with no difference existing between 30 min and 1 h of stimulation in high glucose concentrations.
However, in low glucose concentrations, the amount of insulin was obviously lower than that in high-glucose stimulation for 30 min or 1 h. Interestingly, Cell press normal human pancreatic beta cells responded to low glucose concentrations after 30 min of stimulation, and the amount of insulin was similar to the amount resulting from 1 h of stimulation. On the other hand, IPCs hardly secreted any insulin (0.46 ± 0.04 μU/mL) after low-glucose stimulation for 30 min and only secreted a little insulin (1.01 ± 0.11 μU/mL) after 1 h of stimulation in low glucose concentrations. Our data illustrated that insulin secretion from both normal beta cells and IPCs were regulated by glucose. However, the amount of insulin secreted by beta cells was much higher than that by IPCs (P < 0.05).