Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations

Karsten Burkert,Gabriel Peinkofer,Kurt Pfannkuche,Matteo Oliverio,Sarkawt Hamad,Hadiseh Taheri,Jürgen Hescheler,Albrecht Berkessel,Tomo Šarić,Jan-Wilhelm Kornfeld,Wacharee Harnying

doi:10.1038/s41598-021-81351-z

Abstract

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.

Highlights

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells
In an attempt to reduce the teratogenicity of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) preparations in animal experiments, we found that the previously described inhibitor PluriSIn #136 has a low cytotoxic activity against mPSCs in vitro (Supplementary Fig. S1, Supplementary Results)
Dose–response curves obtained with three different human induced PSCs (hiPSCs) lines (Supplementary Tables S1 and S2) and three different mPSC lines (Table S3) revealed that the most effective salicylic diamines displayed cytotoxic activity against mPSCs with a half maximal inhibitory concentration (IC50) ranging from 0.20 ± 0.17 μM for SM2 and 0.24 ± 0.18 μM for SM6 to 1.1 ± 0.4 μM for SM8 while the I C50 for hiPSCs was in similar range for SM8 (~ 0.4 μM) but much higher for SM2 and SM6 ranging, respectively, from 2.7–11.0 μM and 1.6–12.5 μM for different hiPSC lines (Supplementary Tables S2 and S3, and Fig. S2)