Scarless Enriched selection of Genome edited Human Pluripotent Stem Cells Using Induced Drug Resistance

Abstract

An efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of genome-edited clonal populations. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.

Structure-Activity Relationship Analysis of YM155 for Inducing Selective Cell Death of Human Pluripotent Stem Cells

“…Based on the role of the solute carrier protein SLC35F2 in the import of YM155 (Winter et al, 2014), and differences in the cytotoxicity of YM155 toward different cancer cell types (Winter et al, 2014), we hypothesized that the stemotoxic activity of YM155 and its analogs may be mediated by SLC35F2. To explore this, cell death of hESCs after treatment with various stemotoxic YM155 analogs was compared with control hESCs (wild type: WT) and SLC35F2 knockout (KO) hESCs generated using the CRISPR/CAS9 system (Figures 6A,B and Figure S4; Kim et al, 2019). As predicted, the stemotoxic activity of YM155 and its analogs was completely lost in KO hESCs, while cell death was evident in WT hESCs (Figures 6C,D).…”