Multiplexed targeted high throughput screening for molecules which affect pluripotent stem cell differentiation into pancreatic beta cells

Type 1 diabetes (T1D) is a chronic disease caused by the loss of insulin-producing β-cells, which reside in the pancreatic islets of Langerhans. In-vitro differentiation of human embryonic stem cells (hESCs) into functional β-cells could serve as a source for replacement therapy. However, the cost and low yield of current protocols make this promising therapy out of reach for most patients. While endocrine cells rarely divide, in-vitro differentiation protocols include an intermediate progenitor cell-state which, if maintained, could serve to expand the number of differentiating cells, and dramatically reduce costs. This, however, would require identification of factors which control the progenitor-to-endocrine balance.

We therefore performed a high throughput screen (HTS) for small molecules that affect progenitor-to-endocrine differentiation. We developed a method for RNA-sequencing Of Selected Amplicons (ROSA-HTS) which allows targeted sequencing of up to 250 genes from thousands of samples, simultaneously. Screening 1280 molecules identified 91 factors that affect the transcriptional profile of differentiating hESCs at the stage of progenitor-to-endocrine differentiation, and a validation screen confirmed most of the results. We performed full RNA-sequencing on these “hits” and used previous scRNA-seq analyses to identify expression-programs which correspond with either the progenitor or endocrine state. Notably, we classified the “hit” molecules into groups according to their effect on the differentiation profile.

Combining molecules from different progenitor-promoting groups is expected to have a synergistic effect and improve our ability to maintain self-renewing progenitors. Similarly, combinations of pro-endocrine molecules are expected to improve our ability to push these progenitors to an endocrine fate.