A New Class of Pluripotent Stem Cell Cytotoxic Small Molecules

Abstract

A major concern in Pluripotent Stem Cell (PSC)-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly, cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore, these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo.

Emerging innovation towards safety in the clinical application of ESCs and iPSCs

“…New synthesized small molecules (such as JC011), which selectively induce a cytotoxic endoplasmic reticulum stress response in ESCs and iPSCs, have also been reported, but further studies should reveal the precise mechanisms of this pathway. 12 We believe that two issues relating to the use of ESC or iPSC therapies need to be addressed. After treating cells with chemical inhibitors to prevent teratoma, these cells should be tested to ensure that they have maintained functional properties, including differentiation capacity 13 and engraft ment potential.…”

Dioxonaphthoimidazoliums are Potent and Selective Rogue Stem Cell Clearing Agents with SOX2‐Suppressing Properties

“…A) Small molecules that have been reported to prevent teratoma formation in stem cell cultures. [11][12][13] B) Structures of IV-1 and IV-7,which are potentc ytotoxica nalogues of YM155. [17] Figure 2.…”

“…[10] Thed eploymento fs mall molecules to selectively removeu ndifferentiated pluripotent cells has received attention as av iable alternative. [11][12][13] This pharmacological approach has several advantages:i ti sr obust, rapid, andc ost-effective, maintains genetics tability of cells, minimizesc ell attrition,a nd has good translational potential, as it can be appliedt oc omplex differentiation protocols. Figure 1A shows small molecules that have been reportedt oi nhibit the formation of stem-cellderived teratomas.…”

“…[11] Disruption of this pathway led to ER stress, unfolded protein response, translational attenuation in PSCs, and ultimately,a poptotic cell death.T he N-benzylnonanamide JC011, as tructurala nalogue of capsaicin, also induced ER stress by activating the PERK/AT4/DDIT3 pathway. [12] The flavonoid quercetin and dioxonaphthoimidazoliuma nalogue YM155i nhibited survivin, an anti-apoptotic protein whose gene (BIRC5)i ss trongly linked to teratoma formation. [13,14] The rationalef or targeting survivin rests on ab alance of pro-and anti-apoptotic genes in undifferentiated stem cells.…”

Full biological characterization of human pluripotent stem cells will open the door to translational research

“…A high-throughput inhibitor screen revealed that the SCD1 inhibitor, also called pluripotent cell-specific inhibitor #1 (PluriSIn #1) induces endoplasmic reticulum (ER) stress, inhibits protein synthesis and leads to apoptosis in hESC and hiPSC (Ben-David et al 2013). Similar approaches led to the finding of further inhibitors (JC011, JC010, JC017, JC040) targeting the ER of undifferentiated hESC (Richards et al 2014). Treatment with the DNA topoisomerase inhibitor etoposide results in depletion of undifferentiated hiPSC from cardiac progenitor populations.…”