Novel high throughput screening strategy for the identification of small molecules which affect in vitro beta cell differentiation

Type 1 diabetes (T1D) is a chronic debilitating disease caused by autoimmune destruction of pancreatic beta cells, which affects millions of people worldwide. Transplantation of cadaveric islets (which harbor beta cells) can provide years-long regulation over glucose levels, but donor scarcity limits its feasibility. Recently, several protocols were devised for the directed differentiation of pluripotent stem cells into functional beta cells, offering an unlimited source for transplantable material. To make this treatment widely available it is necessary to improve the protocols’ yield, either by recapitulating recently-discovered aspects of embryonic islet formation, or by gaining better understanding of the in vitro differentiation process itself.

To find small molecules that improve the current protocols we developed a strategy for High Throughput Screening (HTS) based on RNA-sequencing Of Selected Amplicons (ROSA-HTS). Sequencing of ~250 pre-select genes across more than 1,200 treatment conditions, facilitated discovery of nearly 100 candidate molecules which regulate the balance between early progenitors and differentiated endocrine cells. Full RNA sequencing of samples treated with the potential “hits” uncovered distinct patterns of influence by these small molecules. Specifically, we identified molecules which disrupt the progenitor state, and others which drive endocrine differentiation. Surprisingly, we describe a group of small molecules which seem to promote expression of the endocrine initiator NEUOROG3, but fail to advance cells to later stages of differentiation. Thus, this work provides tools to improve existing protocols for directed differentiation of pluripotent cells into pancreatic endocrine cells, and concomitantly sheds light on the basic biological processes that drive it.