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... 2 day as compared with uh ES. No significant difference in insulin concentration was observed when incubation's were carried out at 5. 5 -mmol / l medium glucose concentration (15816 gU/ml, gU/ml, n = 6) or at a 25 -mmol / l ambient medium glucose concentration (146. 2 bn 22. 1 gU/ml, n = 6). (Assay et. al. , 2001) Method 2: As a result of their ability to differentiate into many different cell types, one can dissect the complex network of transcription factor genes regulating tissue-specific gene expression (Odo rico et. al. , 2001). In order to engineer pancreatic islet cells, which are responsible for the production of insulin requires that one possess some knowledge on the development of the pancreas in an embryo.
The pancreas develops from the fusion of the upper duodenal part of the foregut and ventral diverticula. In mice, by embryonic day 9. 5 (E 9. 5) of development, the ventral pancreatic bud begins to migrate backwards and comes into contact, eventually fusing with the dorsal pancreatic bud during the sixth week of development (Polak, et. al. , 2000). The first molecular sign that part of the gut is committed to a pancreatic fate appears at E 8. 5, with the expression of the homeobox protein (PDX- 1), somatostatin trans activating factor- 1 (STF- 1) or insulin upstream factor- 1 (IUF- 1). The pattern of PDX- 1 expression and its ability to stimulate insulin gene transcription suggest that it functions in the maintenance of the ]-cell identity (Soria et.
al. , 2000). During development, ]-cell masses expand by differentiation of precursor cells in the pancreatic duct epithelium. Throughout the development of the fetus, the ]-cells replicate to further increase the cell mass. In adult life, it is possible that both the replication of pre-existing ]-cells and the differentiation from pancreatic duct epithelium take place. Therefore, it is possible to treat and cure diabetes by genetically manipulating the adult precursor (pancreatic duct cell) and transplanting them back into the pancreas of diabetic people (Soria et. al. , 2000).
By altering embryonic stem cells (ES), an insulin secreting cell was derived from mouse ES cells that normalized blood glucose when transplanted into diabetic mice. Adding DNA containing parts of the insulin gene to embryonic cells from mice in a three step method, enabled the cells to differentiate to produce insulin to culture. In the first step, a cell-trapping system, a method in which cardio myocytes or neural precursors (specific cell types used for transplantation) are obtained, was used to obtain an insulin-secreting cell clone from undifferentiated ES cells. Once they were selected, step two took place.
Using cell-lineage selection, an insulin secreting clone was formed from undifferentiated ES cells. The construction of step two allowed the expression of a neomycin selection system (selecting certain DNA containing part of the insulin gene). This chimeric gene was fused to a hygromycine resistance gene to select transfected cells making it resistant to antibiotic drugs. And by undergoing step three, maturation, the cells were grown in the presence of an antibiotic, only those cells that activated the insulin promoter were able to survive. Selected ES cells were then cultured according to standard protocols except in the last stages of its incubation period, they were cultured in bacterial Petri dishes, allowing cells to form aggregates to facilitate subsequent transplantation in the spleen of diabetic mammals. Lowering the glucose concentration during the last incubation stage developed high productive insulin cell clones (Soria et.
al. , 2000; Nat. Inst. Health, 2001). ES-derived insulin-secreting cells were cultured in different conditions, and their insulin content as well as their secretory responses to glucose were assessed. After culture of the cells in the presence of 10 mmol / l nicotinamide and high glucose for 14 days, very low levels of insulin content were found.
However, when the cells were grown in the presence of low glucose (5 mmol / l ), a progressive increase of insulin content was observed after the first day of incubation. Eventually, the low effect of glucose plateaued after 5 days of culture (Figure 2). Results Despite completely different approaches taken in the different researches, results were similar in that both of their insulin productions increased with lower concentrations of glucose. However, the total insulin content of the cloned insulin-secreting cell in the second method was 16. 5 bn 2. 7 ng / gg protein, corresponding to about 90 % of the insulin content of normal mouse islets (Soria et. al. , 2000). In addition to that, it responded to the changes in the glucose level by increasing its production of insulin when glucose levels rose.
In the first method, only 40 - 70 % of insulin stained positively for insulin and an average of 1 - 3 % cells positively stained at maximum density of glucose. Discussion All of the data above obtained from different researches pertaining to different methods of differentiating stem cells into insulin producing cells implies that method two is the better approach of the two in producing insulin more effectively. Despite some of the implications made by the resulting data of the two methods, there are yet other factors that leads one to consider that method two is better. Take for example, the experimental approaches of the two.
Compared to the approaches taken on in method one, those of method two are more similar to the actual differentiation of the early human pancreas. In other words, it resembles the steps of natural insulin-secretion more closely. For example, in the last stages of the incubation period in method two, cells were cultured in bacterial Petri dishes, allowing cells to form aggregates to facilitate subsequent transplantation in the spleen of diabetic animals. Another aspect of method two that is favorable is its cell-lineage selection system which selects only those cells that express a marker gene. By doing this, the insulin-secreting cells derived from mouse embryonic stem cells were able to normalize glucose (tell the difference between high glucose levels and low glucose levels). The cells derived in method one however, didnt have that ability because the resulting, insulin-secreting cells were hand picked from a large group of other cells that had all differentiated at the same time.
The concept of embryonic stem cells is a fairly new concept thus both of these methods can not yet be applied to actual human beings. Both of the methods one and two mentioned injecting their differentiated cells into the pancreas of rodents. Both of the insulin producing cells continued to produce the cells in-vivo once inside the pancreas but it is not yet known if any of them had the potential to reverse the effects of diabetes. Conclusion With steps such as the discovery of embryonic stem cells being able to differentiate into pancreatic ]-cells and other insulin producing cells, a permanent cure for diabetes draws farther away from the future and closer to us. However, one must remember that the study and use of stem cells is still in its infancy and that it may take several years in order for a cure to be made through its concept. Especially in the cure of autoimmune diseases such as diabetes, a permanent cure with the use of stem cells will be difficult and challenging.
It is important however, to consider how far stem cell biology has come up until this point.
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