Abstract Directed differentiation of induced pluripotent stem cells (iPSCs) enables the production of relevant cell types for studies of human biology, disease-modeling, and efforts towards cellular therapy and transplantation. However, the low yield and purity of desired cell types can limit the utility of this approach. Enhancing differentiation purity can require extensive optimization of morphogen treatments, and this can still be ineffective for iPSC lines with biased or aberrant differentiation propensities. To address these limitations, we have developed a new approach for increasing the purity of iPSC directed differentiation cultures called R NA sequencing and A ntisense-assisted D ifferentiation (RAD). We performed trajectory analysis of single cell RNA sequencing during iPSC differentiation into endoderm to identify transcription factors responsible for committing cells to undesired, non-endodermal fates. Specific suppression of these transcription factors using antisense oligonucleotides (ASOs) increased the percentage of endodermal cells by up to 20-fold in 3 different iPSC lines that exhibited poor endoderm differentiation. Moreover, this approach required minimal culture manipulation. Thus, RAD improves the utility of iPSCs for basic and translational studies.

Single Cell Transcriptomics-guided Antisense Treatment Improves Endoderm Differentiation of iPSCs