Exploring Efficient Generation of InsulinProducing Cells from iPSCs, to Enhance Cell Therapy for Type I Diabetes

Abstract

Type I Diabetes Mellitus (TIDM) is one of the immune disorders and results from the loss of cells that make insulin (INS), a hormone that regulates the level and availability of glucose within the body. The most common treatment is exogenous insulin therapy which involves regular insulin injections or the use of an insulin pump. However, this treatment is imperfect and cumbersome, driving researchers to find more effective methods to control blood glucose levels. One potential treatment would involve making INS producing beta cells using pluripotent stem cells and then using these manufactured cells as a beta cell replacement therapy. Making insulin-producing cells in the laboratory involves several stages from stem cells to definitive endoderm, pancreatic endoderm, endocrine precursor, and the last step to mature beta cells. Any improvements in specific steps for the production of PSC derived pancreatic beta cells has the potential to lower the cost of an eventual treatment, making such therapy more widely available. In this project, we examined variations in the differentiation protocols with the ultimate aim of increasing the yield and functionality of the final cell product.

One of the significant findings to emerge from this thesis is, in Chapter 4, we show that SCF addition has the potential to enhance cell proliferation during the stages of early differentiation. A surface receptor for Stem Cell Factor, cKIT, is widely applied to determine endoderm formation in the differentiation process of in vitro pluripotent stem cells. Despite its long-standing relationship with endoderm formation, a potential role for SCF itself during this process has not been systematically addressed. Using a suspension-based differentiation system, we show that the addition of SCF to differentiating cultures of PSCs enhanced the proliferation of early definitive endoderm, marked by the expression of CXCR4 and EPCAM. This effect of SCF was discernable within 2 days of differentiation initiation and coincided with the down-regulation of its receptor, cKIT. SCF treatment appeared not to affect the kinetics of differentiation, with key markers such as MIXL1, SOX17, CXCR4, and EPCAM showing identical expression patterns in untreated and treated cultures. Taken together, our results indicate that SCF addition at an early stage of differentiation will potentially reduce the number of input PSCs required to yield a given amount of endodermal products for the downstream application.

This thesis is organized into six main chapters. Chapter 1 is a literature review concerning the introduction of pluripotent stem cells, Type I diabetes, pancreas development, normal molecules used in vivo differentiation protocol for beta cells generation. Chapter 2 describes the materials and methods of our experiments. Chapter 3 investigates the role of Activin A in definitive endoderm formation, which attaches great importance to adding Activin A from the beginning of differentiation. Chapter 4 shows that SCF promotes the proliferation during definitive endoderm differentiation. Chapter 5 presents images of the final cell product, which successfully show the signals of insulin. Chapter 6 is related to the limitations of previous research and the suggestions for future work.