MAFA and MAFB gene expression profiling and role during directed differentiation of human pluripotent stem cells to insulin producing pancreatic beta cells

International Diabetes Federation reports showed that the prevalence of diabetes in adult Qataris was 14% in 2017 and is estimated to rise to 22% by 2045. Patients with type 1 diabetes (T1D) are treated with insulin injection, whereas, hyperglycemia in patients with type 2 diabetes (T2D) is controlled by diet and oral medications as first line therapies with insulin added only in patients inadequately controlled on those first line therapies. Pancreatic beta cell replacement therapy represents an ideal treatment and/or cure for patients with diabetes. Although whole pancreas, as well as isolated islets of Langerhans, transplantation have been shown to be effective for reversing hyperglycemia (Shapiro et al., 2000, 2006), the inadequate number of available organs limits this option for the majority of diabetic patients. Therefore, alternative approaches are needed, and human pluripotent stem cells (hPSCs) directed towards insulin-secreting beta cells holds promise for clinical use in diabetic patients. To this end, it is necessary to generate functioning (insulin-producing) beta cells to replace the damaged or lost beta cells in diabetic patients. To achieve this, multistep differentiation protocols that recapitulate pancreas development are designed to direct stem cells towards a pancreatic endocrine fate, and eventually to deliver functioning beta cells (Pagliuca et al., 2014; Rezania et al., 2014). Functioning pancreatic beta cells secrete insulin in response to a glucose challenge, termed glucose-stimulated insulin secretion [GSIS], and reports have demonstrated the role of the musculoaponeurotic fibrosarcoma protein A (MAFA) in appropriate GSIS. However, in vitro, MAFA is not expressed in stem cell-derived pancreatic beta cells (Veres et al., 2019). Furthermore, MAFA expression is known to be reduced in beta cells of patients with T2D (Butler et al., 2012). In rodents, MafB is expressed in immature fetal beta cells, and as beta cells matures there is a switch from MafB/insulin-positive cells to MafA/insulin-positive cells(Artner et al., 2010). The expression pattern and role of MafB/MafA in pancreas development and beta cell formation has been studied using animal models. However, recent reports have highlighted the difference in MAFB/MAFA expression pattern in humans(Arda et al., 2016; Li et al., 2016). Therefore, in this proposal, our main goal is to study the expression of MAFB/MAFA in pancreatic beta cells derived from human pluripotent stem cells. Using CRISPR/Cas9, we propose to generate a double reporter pluripotent stem cell line in which the endogenous loci of MAFA and MAFB genes will be targeted to express Enhanced Green Fluorescent and Cherry fluorescent proteins, respectively, from one allele while the second allele will be intact expressing the endogenous MAFA and MAFB proteins. This model will help in studying the expression and role of MAFB/MAFA in beta cell differentiation and maturation both in vitro and in vivo.

Hamad Bin Khalifa University (HBKU)