Pluripotent stem cells, because of their ability to differentiate into any cell type, have been widely advocated as a means of producing a nearly unlimited source of new insulin-producing β cells for the treatment of diabetic diseases. However, while there has been remarkable progress in learning how to direct the differentiation of human embryonic stem (hES) cells towards pancreatic endocrine cell fates, insulin-expressing cells made in this manner are often polyhormonal and lack a normal response to glucose, thereby suggesting a need for a deeper understanding of the gene regulatory networks that are established in a stepwise manner during pancreas development.
My thesis studies explored three main topics, each of which holds potential for the development of improved hES cell directed pancreatic differentiation protocols and the discovery of genes that may specifically affect β cell development. First, we used mice that contained a fluorescent reporter allele and fluorescence-activated cell sorting (FACS) to isolate several discrete pancreatic cell populations which were then analyzed using whole transcriptome sequencing (RNA-Seq). By doing so, we were able to examine the genetic requirement and temporal changes of cells expressing pancreas specific transcription factor 1a (Ptf1a), a marker of the pancreatic multipotent progenitor cells (MPCs) and of acinar-specified cells, during pancreas development. By comparing the transcriptional profiles, we identified five gene clusters, each of which provides insights into the dynamics of gene expression during specific aspects of pancreas development. Second, my studies revealed that Nephrocan, an inhibitor of the TGFβ signaling pathway, was expressed in pancreatic MPCs. Thus, to explore the role of Nepn further, we generated mice containing a single copy insertion of a Nepn-Cherry transgene. Finally, to facilitate the combinatorial sorting of Pdx1- and Ptf1a-expressing cells during early pancreas development, we generated a mouse line expressing a cyan fluorescent protein under control of the endogenous pancreatic and duodenal homeobox 1 (Pdx1) gene.
The research that I have performed is part of a larger project focused on generating and characterizing a series of high quality transcriptional profiles representing key stages in the generation of pancreatic endocrine cells that occur naturally in the mouse. We anticipate that further analysis of the datasets I have generated for specific developmental stages, in combination with similarly generated datasets at other developmental stages, will facilitate identification of signaling pathways and gene clusters essential for formation of functional pancreatic β cells in the mouse, thereby stimulating new hypotheses for identifying pro-β cell signals necessary to direct the differentiation of pluripotent stem cells into pancreatic β cells.