Toxicity of proinflammatory cytokines towards surrogate insulin-producing cells generated from human pluripotent stem cells

• Applicant: Privatdozent Dr. Ortwin Naujok
• Subject Area: Endocrinology, Diabetology, Metabolism
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 329435715

Recent advances in the development of a cell replacement therapy for type 1 diabetes mellitus (T1DM) using pluripotent stem cells (PSC), such as human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, have gathered momentum. Lately data have been published showing that glucose-responsive beta cells can be produced by in vitro differentiation alone using human ES cells as the cell source. These differentiated cells are potentially suitable for a cell-based therapy of T1DM. However, T1DM is characterized by an autoimmune-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. The mediators of this apoptotic destruction are T-effector cells and pro-inflammatory cytokines, especially IL-1beta, TNF-alpha, and IFN-gamma. This autoimmunity persists during life and there is strong evidence from transplantation studies that autoimmunity can reoccur and destroy any insulin-producing cell transplanted into a patient with T1DM. Thus, before ES cell-based therapies can be translated from experimental studies towards pre-clinical tests it must be determined whether surrogate cells made from ES cells harbor a significant sensitivity towards pro-inflammatory cytokines. Cell death induced by cell-to-cell contacts between cytotoxic T-cells and beta cells is another way by which they can be destroyed but cell-based therapies of diabetes using ES cells envisage encapsulation so that the solid immune system is separated from the graft inside the capsule. Of note, however, those encapsulation devices will not keep out cytokines due to their size and solubility.It is the aim of this project to generate glucose-responsive insulin-producing cells from PSCs in suspension and adherence. Terminally differentiated cells will be functionally characterized in vitro and in vivo. Then, the cells will be analyzed with respect to the expression of cytokine receptors and whether they can amplify autoimmunity by their own expression of chemokines and cytokines. We will address whether the exposure to pro-inflammatory cytokines results in a loss of viability and induction of apoptosis specifically in insulin-producing surrogate cells. This includes also the characterization of the main cytokine-activated signaling pathways, pro- and anti-apoptotic effector protein expression, and the possible interaction between the endoplasmic reticulum and mitochondria. Data gathered from this project will yield the answer by which protective measure surrogate cells may be protected in the future against soluble immune effectors. This could be achieved e.g. by CRISP/Cas9 mediated genomic editing to engineer cytokine-insensitive PSCs. Moreover, the results from this application can serve as a predictor for the success of PSC-based therapies of T1DM in the future.

• DFG Programme: Research Grants