Insulin Secreting Cell In The Second Method example essay topic

Introduction Type 1 Diabetes mellitus, formerly known as insulin-dependent diabetes mellitus is a disease that is defied as a metabolism disorder. It affects about 5-10% of the diabetic population estimating to about 4.9 people worldwide. In this type of diabetes, the onset of elevated blood sugar levels usually begin abruptly in a fairly dramatic way before the age of 30 and about half of all the cases appear during childhood. The cause of diabetes type 1 is an autoimmune destruction in which the immune system produces antibodies that attack the pancreatic lb]-cells. Insulin in the body that serves to suppress glucose production in the liver and its release from storage depots into the bloodstream. Without insulin, glucose in the blood remains virtually useless and the bodies cells are deprived of fuel, despite an increase in blood sugar levels (Alterman, 2000).

The only possible type of treatment of Type 1 diabetes up until recently is taking daily insulin injections while constantly monitoring one's blood sugar level. The only permanent cure available for it is cell replacement therapy (Assady et. al., 2000). However, the lack of suitable donors opposed a major problem in accomplishing it. It wasn! |t until after the discovery of methods to isolate and grow human embryonic stem cells in 1998 by Professor James A. Thomson from the Univ. of Wisconsin that a feasible method came into view. Embryonic stem cells are derived from the inner cells mass of one of the earliest stages in the development of the embryo, the stage when it is a blastocyst.

Blastocyst's have the potential to self-replicate and is pluripotent (can give rise to cells derived to form all three germ layers), thus being able to differentiate into insulin producing pancreatic cells. Since type 1 diabetes is an autoimmune disease, the use of stem cells to restore those destroyed cells would be reasonable. The embryonic stem cells of working pancreatic islets cells are extracted from a rodent (due to controversial issues dealing with the use of human embryonic stem cells) and cultured on mediums until they differentiate. They are then implanted into a person or another rodent with diabetes so that they would function in-vivo to process the glucose like an actual pancreatic islet cell would. Two Different Approaches to Stem Cell Differentiation In order to successfully differentiate stem cells to replicate normal insulin producing cells of the pancreas, several criteria must be met. Most importantly, stem cells should be able to multiply in culture and reproduce themselves exactly.

They should also be able to differentiate in vivo to produce the desired kind of cell (Nat. Inst. Health, 2001). The level of success can be measured at the end of each research by using a highly sensitive radio immunoassay (a device that is used to measure the insulin and glucagons concentrations in culture media) to determine how much insulin the differentiated cells produce in the produce in the presence of a certain amount of glucose (Zulewski et. al., 2001). In this paper, two different methods of differentiating stem cells are investigated. The first method is a five-step culturing method which involves inducing mouse embryonic stem cells to differentiate into insulin-secreting structures that resemble pancreatic islets.

The second method is an engineered differentiation approach also known as a three-step-method consisting of directed differentiation, cell-lineage selection, and maturation. This method involves the use of genes to create insulin producing lb]-cells.! K... Method 1: When removed from their normal embryonic environment and cultured under appropriate conditions, inner cell masses give rise to cells that proliferate and replace themselves indefinitely. Yet while in this undifferentiated state in culture, they maintain the developmental potential to form advance derivatives of all three e.g. layers (Odorico et. al., 2001). The five-step culturing method is one in which, an embryonic stem cell is left to form embryoid bodies, and a population of cells expressing the neural marker nestin is selected. Nestin is a protein specifically expressed in the neural stem cells of the brain.

They are present in the neural tube of the developing rat embryos at embryonic day 11 and have phenotypic similarities between embryonic islet cells, giving rise to any type of cell like the pancreatic islet. Because cells that are derived from islets can differentiate in culture into cells of the pancreas, they are able to secrete detectable levels of islet hormones such as insulin (Zulewski et. al., 2001). Human embryonic stem cells grew as homogeneous and undifferentiated colonies when they were propagated on a feeder layer of mouse embryonic fibroblasts (MEFs). A tissue containing large stocks of primary MEFs was then prepared and stored in liquid nitrogen. After each thaw, cells were used for only 3-5 passages (Assady et al., 2001). The Human embryonic stem cells (hES) were then maintained in the undifferentiated state in culture on a feeder layer of mitotically inactivated MEFs on gelatin-coated six-well plates.

When removed from feeder layers and transferred to suspension culture, ES cells begin to differentiate into multicellular aggregates of differentiated and undifferentiated cells, termed embryoid bodies (EBs) which resemble early post-implantation embryos (Odorico et. al., 2001). Therefore, when ~107 undifferentiated hES cells were disaggregated and cultured in suspension in 100-mm bacterial-grade petri dishes a synchronous differentiation characterized by initial formation of small aggregates, followed by the formation of embryoid bodies resulted. These cells were left unpassaged until confluence (roughly 10 days) and were related on gelatinized six-well tissue culture plates without the feeder layer. This led the cells to spontaneously differentiate to an array of cell phenotypes (Assady et. al.

2001). Embryoid bodies that resulted from the differentiation were then collected and washed three times with ice-cold phosphate-buffered saline, fixed overnight in 10% neutral-buffered formalin, dehydrated in graduated alcohol, and embedded in paraffin. To detect the amount of insulin produced in the different cells, MEFs undifferentiated hES cells, and cells that had differentiated spontaneously in vitro for more than 20 days were grown in six-well plates. To characterize the insulin-containing cells, which were interspersed among the mixed population of spontaneously differentiating adherent hES, insulin elaborated into the medium was measured using the enzyme detecting immunoassay's in undifferentiated hES, differentiated hES, and MEF cells. They were measured at various glucose concentrations and growth conditions. Undifferentiated hES cells (uhES) were cultured in knockout medium (n = 6) or were allowed to differentiated in high-density adherent conditions (hes) for 22 (n = 12) and 31 days (n = 7).

In the undifferentiated hES, an insignificant amount of insulin could be detected (5.6 "bfn 0.6 lbgU / ml, n = 6). However, in the media harvested after 22 and 31 days of differentiation, insulin concentrations were as follows: 126.2 "b 17.7 lbgU / ml (n = 12) and 315.9 "bfn 47 lbgU / ml (n = 17), respectively (Figure 1 a). HES growing in suspension as EBs for 20-22 days (n = 6). Cultures were exposed to 3 ml of serum-free medium for two hours either containing 25 or 5.5 mmol / l glucose. Insulin release was significantly greater from 20 to 22 day as compared with uhES. No significant difference in insulin concentration was observed when incubation were carried out at 5.5-mmol / l medium glucose concentration (15816 lbgU / ml, lbgU / ml, n = 6) or at a 25-mmol / l ambient medium glucose concentration (146.2 "bfn 22.1 lbgU / ml, n = 6).

(Assady 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 (Odorico 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 (IF-1). The pattern of PDX-1 expression and its ability to stimulate insulin gene transcription suggest that it functions in the maintenance of the lb]-cell identity (Soria et. al., 2000).

During development, lb]-cell masses expand by differentiation of precursor cells in the pancreatic duct epithelium. Throughout the development of the fetus, the lb]-cells replicate to further increase the cell mass. In adult life, it is possible that both the replication of pre-existing lb]-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 "bfn 2.7 ng / lbgu 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, didn! |t have that ability because the resulting, insulin-secreting cells were! SSh and 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 lb]-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.