Introduction
Currently, research studies indicate that diabetes mellitus (type 1 diabetes) is one of the leading causes of deaths throughout the world, threatening human lives. Statistics indicate that the number of people living with diabetes mellitus is about 370 million, with an annual increase of over 60% in developed countries and 20% in the developing world. It is estimated that the number of people living with the condition will be more than 550 million by 2030.
Destruction of beta cells (β-cells) in the islets is the cause of a decrease in the volume of insulin released from the pancreas (Amour et al. 1392). Studies have shown that an autoimmune-mediated process is the principal cause of the condition (Lakey, Mirbolooki, and Shapiro 78). Specifically, the autoimmune-mediated process is responsible for a TID etiology, the principle aspect of the β-cell death (Soria, Skoudy, and Martin 407). In this case, antigen-presenting cells infiltrate the islets of Langerhans, attracting CD8 and CD4 T cells, B cells, and NK cells. Macrophages are responsible for producing IL-12, which activates Th1 CD4 T cells. In turn, the activated CD4 cells release IL-2 and pro-inflammatory cytokines, TNF, and interferon-γ, leading to activation of CD8 T cells (Reubinoff et al. 400). This enhances the death of β-cells through apoptosis.
Principle
There is evidence that transplants of islets of Langerhans with β-cells can reverse the death of these cells in the pancreas to counter diabetes mellitus (Kroon et al. 458). Human embryonic stem cells have been directed to proliferate and develop into β-cells (Thomson et al. 1146). It is, therefore, expected that induced pluripotent stem cells (iPS) can be directed in the same manner. In this method, the iPS stem cell line will be developed as directed to become full β-cells for placement in human kidneys to generate insulin (Odorico, Kaufman, and Thomson 193).
Study problem
Despite the presence of knowledge, few studies have produced effective cell lines because the problem is to generate an adequate source of these cells in practice and research (Sams and Powers 83).
Materials and methods
The study will work with human iPSCs line RSCB0082 to be purchased from a stem cell bank. The cells will be reproduced from reprogramming human dermal fibrobrast cells. They will be grown in a cell medium with mitotically inactivated rat or mouse embryonic fibroblasts and 70% Gibco medium. Gibco Medium has 20% knockout serum, 0.1mM of amino acids, mercaptoethanol, transferrin, penicillin, and streptomycin (Cowan et al. 1355).
Procedure
The method will involve four steps that will lead to the development of mass iPCs with the capacity to reverse diabetes mellitus.
Step 1
After obtaining the cells, the colonies will be trypsinized with an agent such as EDTA. A single-cell suspension will be developed through sipping cells gently in an up and down manner using a pipette. Then, the cells will be counted. Hanging drops will be made using 30µl, each with about 3000 cells in a media high in DMEM and 10% fetal bovine serum but deficient in bFGF (Bonner-Weir and Weir 853). Then, the cells will be placed on the lids of Petri dishes and incubated for 72 hours.
Step 2
After 72 hours, the embryonic bodies of cells will be plated again in 6-well culture plates and a density of 100 bodies per well (Chen et al. 266). The culture plates will be coated with gelatin (about 0.1%) and incubated for 14 days in a DMEM medium with insulin, selenium, and transferrin (Hwang et al. 1672). They will be incubated for 7 days
Step 3
After incubation, the cells will be separated with EDTA rich in 0.05% trypsin. They will be placed on 6-well cultures at a concentration of about 300,000 per well. The wells will be supplied with DMEM media-rich in Nitrogen 2 supplement (1%), B-2 supplement (3%), and fibroblast growth factor (FbGF). In addition, the culture wells in the plates will be coated with sigma gelatin of about 0.2%. It is expected that after 7 days, cell clusters will be formed on the plates (Murtaugh 429).
Step 4
Before removing the fibroblast growth factor, the cells will be supplied with 1% and 2% of N-2 and B-27, respectively. After the removal of the fibroblast growth factor, nicotinic acid will be added to the cultures (Jiang et al. 1948).
Quantification processes
Quantification of a pancreatic specific factor of transcription in the cell lines will be done using a real-time polymerized chain reaction (PCR). The purpose is to determine the amount of RNA produced from the cells because it is expected that after the four steps above, they will produce mRNAs.
The secretion of insulin in the cell lines will be investigated through a final analysis involving the centrifugation of some cells and radioimmunoassay of their supernatants. In this case, some clusters of the cell lines will be rinsed with Krebs-Ringer buffer (HEPES) rich in glucose. The cell line clusters will be incubated in this medium for 10 minutes, 20 minutes, 30 minutes, 50 minutes, and 60 minutes in the buffer. Then centrifugation of each of the cells will be done, and a radioimmunoassay did on the supernatant to determine the presence of insulin protein from the cell lines (Maehr et al. 15769).
Statistical analysis with SPSS or ANOVA will be used to develop and analyze data from each of the 6 sets of radioimmunoassay tests.
Expected results
It is expected that iPCS will differentiate into ILCs using this protocol. It is also expected that the cells will produce endocrine progenitors, which will then be transmitted into the kidney of a mouse model deficient in insulin to observe their possible effects on humans.
Works Cited
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