Exploring the use of human pluripotent stem cells to create functional pancreatic beta cells

Directed differentiation of human pluripotent stem cells (hPSCs) has the potential to produce human cell types that can be used for disease modeling and cell transplantation. Two key challenges in the differentiation from hPSCs to beta cells are the specification from pancreatic progenitors to insulin-expressing (INS+) cells and the maturation of INS+ cells into glucose responsive beta cells.;To address the first, two high-content chemical screens identified PKC inhibitors as inducers of INS+ cells from pancreatic progenitors. PKC inhibition generated up to ten-fold more INS+ cells while PKC agonists blocked differentiation into INS+ cells. Transplantation of PKCbeta inhibitor-treated pancreatic progenitors, containing higher proportions of endocrine progenitors and endocrine cells, resulted in mature beta cells showing higher levels of glucose-stimulated human c-peptide production in vivo. This indicates that in vitro derived INS + cells might be competent to mature into functional beta cells.;To address the second challenge, we first studied mouse and human beta cell maturation in vivo. Postnatal mouse beta cell maturation was marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3. To study human beta cell maturation, a Method for Analyzing RNA following Intracellular Sorting (MARIS) was developed and used for transcriptional profiling of sorted human fetal and adult beta cells. Surprisingly, transcriptional differences between human fetal and adult beta cells did not resemble differences between mouse fetal and adult beta cells, calling into question inter-species homology at the late stages of development.;A direct comparison between hPSC-derived INS+ cells, and beta cells produced during human development is essential to validate directed differentiation and provide a roadmap for maturation of hPSC-derived INS + cells. Genome-wide transcriptional analysis of sorted INS + cells derived from three hPSC-lines suggest that different lines produce highly similar INS+ cells, confirming robustness of directed differentiation protocols. Furthermore, non- functional hPSC-derived INS+ cells resemble human fetal beta cells, which are distinct from adult beta cells. We therefore suggest that in vitro directed differentiation mimics normal human development and reveal differences in gene expression that may account for the functional differences between hPSC-derived INS+ cells and true beta cells.