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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 ]-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 ones 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 wasnt 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. Blastocysts have the potential to self-replicate and is plenipotent (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 ]-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 EG layers (Odorico et. al. , 2001). The five-step culturing method is one in which, an embryonic stem cell is left to form embryo bodies, and a population of cells expressing the neural marker nesting is selected. Nesting 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 embryo 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 embryo bodies resulted.
These cells were left unassuaged 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 (uh ES) were cultured in knockout medium (n = 6) or were allowed to differentiated in high-density adherent conditions (dh ES) for 22 (n = 12) and 31 days (n = 7). In the undifferentiated hES, an insignificant amount of insulin could be detected (5. 6 bn 0. 6 gU/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 gU/ml (n = 12) and 315. 9 bn 47 gU/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 2...
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