Isolation And Identification Of Pancreatic Stem Cells In Neonatal Piglets

Diabetes mellitus is the syndrome caused by hyperglycosemia. With the increasing morbidity of diabetes mellitus, the diabetes mellitus has now become the third highest incidence following cardiovascular disease and tumour which are chronic diseases heavily threatening people's common health. The cardinal principle of diabetes therapy is to supplied with insulin rapidly, availably and chronically to replenish the self-secreting lack of insulin. There are 3 kinds of methods to be used: ①the traditional methods of injecting exogenous insulin hypodermic were effective. However, the disadvantages of them limited their uses, which the supply need to be inconsistent with the demand of the body, and the injection every day and every dinner is somewhat harassment. Further more, they can not prevent the occurring and developing of severe complication. ②The transplants of pancreas or islets can solve the inconsistency of supply and demand, but the complex surgical brings many adverse effects. Transplant rejection could reject the grafts. ③The stem cells implant can redeem the disadvantages of above two methods, in which the stem cells can be easily and simply infused into the body becoming self-pancreas with persistent effect by once administer. The transplanted stem cells could regulate the ratio of cells secreting insulin and related cytokines on the basis of the physiological requirement to avoid the syndrome, lower immunological rejection. The high proliferation rate of them can offer enormous cell-seeds. But the application of stem cells is restricted by the huge-gap lack of donor-tissues. The best xenogenic-animal surrogate is the pancreatic stem cells from neonatal piglets, it has many advantages including the unlimited donor-tissue source for its high breeding rate, similar structure and bioactive of pig-derived insulin with that of human being, approximative blood-glucose regulation-point with human being, and the fact that it can survive in human serum with no harm to the body. Therefore, the study on pancreatic stem cells of neonatal piglets is becoming the promising cure of diabetes. In order to offer the basic information to the clinical application of pig pancreatic stem cells, this dissertation focuses, in three aspects, on the isolation, culture of the neonatal porcine pancreatic stem/progenitor cells, and also the determination of bio-characteristics and the in vitro differentiation of them into various tissues. 1. The establishment of the optimal method for isolating piglet pancreatic cells and the culture system. The fresh pancreatic tissue, collected surgically from the living piglets, was chipped into small fragments after all the lipid tissue and vasculars were clipped on the ice, and digested by 0.25% trypsin into small cell-clusters and single cells. Then filtered and centrifuged three times at lower speed or via density gradient by 5% bovine serum albumin (BSA), through which the pancreatic cells were collected, and cultured in the Petri dish or flask coated with coated by laminin or gelatin, using the lower-glucose DMED supplemented with 10% neonatal bovine serum(NBS) and 10 mmol/L nicotinamide as basic medium. 2 days later, the none adhered(floating) cells were removed, the lower-glucose DMEM supplemented 10 ng/mL epidermal growth factor(EGF) instead of nicotinamide was used as the medium to enhance the proliferation of the pancreas stem/progenitor cells. The results showed that the progenitor cells with endocrine activity presented as multisynaptic cells. 2. The characteristics of pancreatic stem cells from piglets. Primary cells adhered to dishes in form of single cell or cell mass. During the culture, there are some cells which departed from the cell mass continually. Until 7 day's culture, there appeared the multisynaptic-endocrine progenitor cells among the remained cells and cell mass, developing into clone-like, and quickly into jointed reticular form. The number of small cells increased thereafter, while the ductal epithelium-like stem cells proliferated much slowly. The data observed by electron microscopy(EM) show that the characteristic of multisynaptic cells is long synapse, polarization, with big nucleo-cytoplasmic ratio and the immature organelle. The characteristic of small cells presents a great number of micromilli, 10~12μm diameter, insulin-positive. The epithelium-like cells are round, at big nucleo-cytoplasmic ratio, the progenitor with organelles and petty secretory granules. On the whole, three kinds of different cells with stem cell properties were isolated successfully. The immunohistochemical staining showed that the isolated cells express not only the markers of stem cell including SSEA-1, SSEA-4 and Oct4, but the pancreatic cells including PDX-1 and CK19 as well, which indicated that the cells isolated possess the properties of pancreas stem cells. The growth curve of pancreatic stem cell in neonatal piglets is performed. The isolated cells, which have transferred of culture to fourth passage hitherto, can be transferred to serial subcultivation after 12~15 day's culture in vitro. Succeed frozen and thawed had preformed and the viability of the thawed cells was more than 90%. Now, 2.5×107 cells were stored by liquid nitrogen cryopreservation. 3. The differentiation properties of the pancreatic stem cells 1). The medium of D-glucose (2 000 mg/L) RPMI 1640 supplemented 10 mmol/L nicotinamide and 5 ng/mL hepatic growth factor(HGF) was used to promote the cell differentiation toward endocrine ?-cell of islets. Through dithizone (DTZ) staining whichselectively stains pancreatic ?-cell crimson red, the induced cells gradually showed insulin-positive. The average quantity of insulin stimulated with 22 mmol/L glucose was 1.84 times as that with 5 mmol/L, whereas, the average quantity stimulated with 30 mmol/L glucose was lower than that with 5mmol/L. The results above suggested that the induced cells have the function of secreting insulin as ?-cell, the quantity of the secreted insulin varied with the concentration of glucose. The hypo-concentration of glucose may have toxic effect on the cells resulting in lower secretion of insulin instead. 2). The isolated pancreatic stem cells would differentiate toward myocardium exposed to 10 μmol/L 5-Azacitidine for 24h, the cells became longer in shape, expressed a-actin myocardial protein, and presented positive of periodic acid Schiff reaction(PAS). 3). Prior to treating with medium supplemented 5% NBS and 1 mmol/L ?-mercaptoethanol (Me) for 24h, the isolated cells were treated with 5 mmol/L ?-Me, no serum for 4h sequentia to differentiate toward neural cells. The induced cells expressed the marker of neural cells, including NF, GAFP and BMP. 4). Differentiation of isolated cells toward the osteoblast lineage can be induced by supplementing 0.1 μmol/L dexamethasone, 10 mmol/L ?-glycerophosphate and 50 μg/mL ascorbic acid. The round-shaped and cubical-shaped cells were increasing in induced cells forming cluster after 20 day's culture. The cells stained with Alizarin Red were typical bright red, and were positive stained with alkaline phosphatase (AKP). For the first time, multipotent capability of pancreatic stem cells in vitro was studied. The results suggested that the isolated stem cells from piglets pancreas had the multipotent capability of stem cells.