Identification of cell source after Percoll discontinuous density centrifugation fter Percoll discontinuous density centrifugation, the pancreatic tissue cells were distributed at three different densities (D-Hank’s/1.068, 1.068/1.096, 1.096/1.118) on the first, second and third density interfaces. Dithizone staining results showed that 80%-90% of the cells collected from the first and second density interfaces were iron red (Figure 1), indicating that the cells were derived from pancreatic endocrine portion (i.e., pancreas islet). Cells collected from the third density interface were non-stained with dithizone, indicating that the cells were derived from the exocrine portion of the pancreas.     Primary culture and identification of pancreatic stem cells derived from pancreatic islets After 48 hours primary culture, from the pancreatic endocrine there were some large, round, and single-nucleated cells surrounding islet-like cell masses. These cells had strong cytoplasmic refraction, a large proportion of caryoplasm and grew adherently to the wall (Figure 2). These cells reflected the morphology characteristics of pancreatic stem cells, which were confirmed as pancreatic stem-like cells.   The expression of Nestin in pancreatic stem cells was detected by immunocytochemical staining and got positive result as the cytoplasm was stained claybank. These confirmed the pancreatic stem-like cells were confirmed as islet-derived pancreatic stem cells (Figure 3).   The pancreatic stem cells derived from pancreatic islets were continuously passaged to establish cell lines under feeder layer conditions The feeder layer was prepared from mouse embryonic desmocytes. Then, the pancreatic stem cells were subcultured in the feeder layer. After 48 hours of subculture, the pancreatic stem cells began to differentiate and proliferate and form large colonies (Figure 4A). After 96 hours of passage, the pancreatic stem cells could grow to more than 80% of the orifice area (Figure 4B). Then, the cells could be passaged continuously. Under stable in vitro conditions and with mature operation technique, pancreatic stem cells were passaged till the 30th generation to established the cell strain.   Islet-derived pancreatic stem cell line detection Pancreatic stem cells in the process of continuous passages showed an active ability of division and proliferation (Figure 5). Pancreatic stem cell lines still maintained the original morphological features and expressed Nestin during the continuous passage. Under the microscope, the 15th and the 30th generations of pancreatic stem cells had large, round, single nucleus, large ratio of cytoplasm, and strong proliferating ability (Figure 6). Nestin staining showed positive results of all generations of pancreatic stem cells, while negative cells were found in the surrounding feeder layers (Figure 7).  After continuous induction and differentiation, a part of cells began to gather, but no significant changes were found in cell morphology. These cells were divided to produce more cells budding to grow upward, forming a spherical islet-like cell structure after about 1 month, having spindly pedicles connected with the bottom of the bottle, and floating in the culture medium. Dithizone staining was iron red (Figure 8).   The P53 gene of synthetic cDNA was amplified by RT-PCR, and the amplified band was about 763 bp by 1.0% agarose gel electrophoresis. This result indicated that the reverse transcription product cDNA had good quality. PCR was carried out using the specific primers of the insulin gene and the cDNA of the islet-like cell cluster as the template. The target band was found by 1.0% agarose gel electrophoresis. The molecular weight of PCR products was 50 bp in consistence with the expected molecular weight of the insulin gene (Figure 9). However, before induction, the cells did not express insulin mRNA."

"