EFFECT OF METHIONINE DEPRIVATION IN SCALABLE DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS TO β CELLS
<p align="justify">Diabetes mellitus is one of the diseases that affects people world wide. Transplantation of human islets from cadaveric donor has been performed for years to undertake this problem. However, this method has many obstacles such as limited source of donors and nonhom...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/28167 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | <p align="justify">Diabetes mellitus is one of the diseases that affects people world wide. Transplantation of human islets from cadaveric donor has been performed for years to undertake this problem. However, this method has many obstacles such as limited source of donors and nonhomogeneous islet properties. One of the tools to overcome this problem is using iPSC-based cell therapeutics particularly using induced pluripotent stem cell derived-β cells (sc-β). Induced pluripotent stem cells or iPSCs are cells that are derived from somatic cells and reprogrammed into their pluripotent state. These cells can differentiate into three germ layers, endoderm, mesoderm, and ectoderm derivatives thus can be differentiated into pancreatic β cells and to overcome the stated problem. This differentiation process occurs in a stepwise manner. iPSCs can gradually differentiate into definitive endoderm (SOX17+, stage 1), to primitive gut tube (HNF1β+/HNF4α+, stage 2), to pancreatic progenitor (PDX1+, stage 3), to endocrine (NKX6.1+, stage 4), and to β cell (PDX1+/INS+) as the final product. Several successfull researches had been conducted regarding sc-β. However, these researches were mainly conducted in Two-dimensional culture system, hence clinical trial can hardly be performed due to low cell number. Three-dimensional (3D) culture system with an optimum hydrodynamic system can produce higher number of cells. Additionaly, deprivation of one of the essential amino acids, methionine, is known to be required by iPSCs to differentiate to β cells in higher efficiency. The aim of this study is to investigate the effect of methionine deprivation treatment in the upscaled differentiation culture of iPSCs to β cells using spinner flask. In this study, the effect of methionine deprivation treatment (ΔMet) was observed in 3D culture system to allow higher cell number production using hydrodynamic system in spinner flask. This experiment was compared with small scale culture in 6 well plate. Cells in spheroid form were being induced to differentiate using 5-staged process. Spheroids in each stage were cultured with different kinds of medium which contain different growth factors, cytokines, and small molecules that gradually induce iPSC from stage 1 to 5. In the end of stage 1, stage 3, and stage 5, spheroids were analysed using Immunocytochemistry to identify cell differentiation efficiency. In the end of stage 5, Glucose Stimulated Insulin Secretion (GSIS) was performed to identify sc-β response towards glucose stimulation. The results showed that in the upscaled culture, hydrodynamic system inside the spinner is crucial. In the end of stage 1, percentage of cells that differentiated into definitive endoderm cells (SOX17+ cells) are 91.14%, 95.46%, and 93% in upscaled complete culture, upscaled ΔMet culture, and in a small scale culture in 6 well plate culture respectively. In the end of stage 3, we identified pancreatic progenitor cells (PDX1+ cells) percentage. The highest cell percentage was found in 6 well plate culture with 93%, followed by 78.91% in upscaled complete and 72.60% in upscaled ΔMet culture. In the end of stage 5, we successfully generated sc-β (PDX+/INS+ cells) with the highest cells percentage reaching 67.49% in the complete upscaled culture. Upscaled ΔMet culture did not show higher spheroids differentiation into PDX+/INS+ compared to complete upscale culture. Upscaled ΔMet only reached 30.50% while the upscaled complete culture showed 45.67%. GSIS result showed that spheroids in upscaled culture responded to glucose stimulation, however not sensitive to glucose concentration change. In summary, we successfully generated sc-β in higher scale with PDX+/INS+ spheroids percentage reaching 67.49%. However, methionine deprivation treatment in upscaled production did not increase differentiation efficiency of iPSCs into β cells.<p align="justify"> |
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