Differentiation of Human Cells and Tissues Using a Comprehensive Human Transcription Factor Library

Abstract

Functional cells and tissues differentiated from human pluripotent stem cells (hPSCs) have potential applications for disease modeling, drug discovery and regenerative therapy. Current differentiation methods, however, remain complex and time-consuming. For the purpose of rapid generation of pure population of cells, protocols typically result in heterogenous mixtures with low yields. In the case of organoid tissue differentiation, key cell types are missing. Recently, transcription factor (TF)-mediated conversion of hPSCs into differentiated cells and tissues have improved these differentiation methods. First, in order to systematically explore the capacity of TF-mediated cell conversion, we created the human TFome expression library, the first comprehensive human TF library of over 1,700 open reading frames (ORFs) representing 1,576 TFs. We screened the human TFome to identify TFs that could individually induce loss of pluripotency in hiPSCs. We found 243 hits that could differentiate multiple hiPSC lines, even in pluripotency-reinforcing conditions after only four days. This suggests a widespread ability for individual TFs to induce differentiation. Twenty-five top ranking hits were validated and preliminary characterization suggests they induce diverse cell fates. Lastly, we present two novel cell conversions from hiPSCs into functional neurons and stromal fibroblast cells with nearly complete conversion efficiency. Together, these results illustrate the power of the human TFome library to produce many differentiated cell types with high efficiency and speed. We then applied TF-mediated cell conversion to improve organoid tissue differentiation. The interior of many organoids are necrotic due to a lack of vasculature. Previous attempts to vascularize cerebral organoids with human umbilical vein endothelial cells (HUVECs) resulted in their spontaneous separation away from developing organoids. Here, we devised a TF- mediated strategy where alternative fates are induced in the same media conditions. We identified a splice isoform of ETV2 that potently converts hiPSCs into functional endothelial cells even in neural-inducing conditions. This enabled simultaneous production of TF-driven endothelial cells alongside media-driven neural cells and led to the vascularization of cerebral organoids. This hybrid differentiation approach could be generalized for the incorporation of missing cell types in other organoids and tissues.