A new class of pluripotent stem cell cytotoxic small molecules

10.1371/journal.pone.0085039PLoS ONE93-POLN

JC011 treated PSCs fail to form teratomas in SCID mice and can be effectively used to enrich for specialized cells.

<p>Teratoma sections from SCID mice. Controls showing typical teratoma tissue organization representative of all 3 germ layers, A = Cartilage, B = Gut/Intestine, C, D = JC11 treated sample showing plain muscle tissue with no teratoma growth (<b>A–D</b>). BGO1V and primary neonatal cardiomyocytes were mixed and seeded in pre-determined ratios (10∶90, 20∶80, 30∶70, 60∶40 and 50∶50). The mixed cultures were treated with JC011 at 20 µM for 12 hrs followed by SSEA-4 and TRA-1-60 FACS analysis to determine enrichment ratios (<b>E</b>). Time course cell viability data for JC011 (20 µM) treated primary neonatal cardiomyocytes indicate that cardiomyocytes maintain high cell viability (>95%) even after a 5-day incubation period with JC011 (<b>F</b>).</p

Table of structure, molecular mass, ORAC and DPPH antioxidant data for 6 JC analogues (JC005, JC007, JC010, JC011, JC017 and JC040).

<p>Table of structure, molecular mass, ORAC and DPPH antioxidant data for 6 JC analogues (JC005, JC007, JC010, JC011, JC017 and JC040).</p

siRNA knockdown of the key ER stress genes ATF-4 and DDIT3 leads to reduced sensitivity towards JC011 in NCCIT cells.

<p>ATF-4 and DDIT3 were silenced via siRNA knockdown. Sensitivity towards JC011 was attenuated in DDIT3 knockdown and ATF-4 knockdown (P<0.05) NCCIT cells thereby confirming involvement of the PERK/ATF4/DDIT3 ER stress pathway in JC011 mediated cytotoxicity (<b>A–E</b>). qRT-PCR confirmation of ATF-4 and DDIT3 transcript knockdown (<b>F</b>). Cell viability figures were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3). Statistical analysis was performed with the Student's T-test.</p

Dose response cytotoxicity data.

<p>Dose response curves for 3 PSC cell lines (BGO1V, H9 and iPS-Foreskin-1) following treatment with JC011 (<b>A</b>). Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011 (<b>B</b>). Time course cytotoxicity analysis for differentiating BGO1V cultures treated with JC011 at 4-day intervals following bFGF withdrawal (<b>C</b>). Dose response curves for 3 JC011 analogues (JC005, JC017 and JC040) showing that JC040 with a longer alkyl side-chain is the most potent analogue (<b>D</b>). Cell viability values were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3).</p

Comparative microarray analysis reveals involvement of the PERK/ATF4/DDIT3 ER stress pathways.

<p>Clustering of key differentially upregulated ER stress genes in BGO1V following 6 µM JC011 (<b>A</b>). Top 10 components of the PERK/ATF4/DDIT3 ER stress pathway that were found to be rapidly upregulated in JC011 treated BGO1V cells (<b>B</b>). qRT-PCR confirmation of upregulated UPR/ER stress pathway genes following JC011 treatment (<b>C</b>).</p

JC011 and JC040 reduce ROS levels in PSCs.

<p>Cytotoxic JC011 and JC040 induce a small but rapid reduction of intracellular ROS levels in BGO1V cells as confirmed by DCHF-DA FACS analysis 3 hrs after treatment (<b>A</b>). JC007 (non-cytotoxic) does not alter endogenous ROS levels while JC005 (non-cytotoxic) increases ROS levels but without any corresponding cytotoxicity to BGO1V (<b>B</b>). FACS histograms are representative outcomes of 4 independent experiments. DCHF-DA stained JC011 treated BGO1V cells (<b>C, D</b>). DCHF-DA stained JC005 treated BGO1V cells (<b>E, F</b>).</p

Morphology of BGO1V-MEF cultures following treatment with 20 mM JC011 for 12 hrs.

<p>“Hollowing-out” effect in BGO1V-MEF cultures, feeders remain intact and viable (<b>A–D</b>). Trypan blue staining of untreated control BGO1V single cells (<b>E</b>). Trypan blue staining of JC011 treated BGO1V single cells showing an increase in Trypan blue uptake (<b>F</b>). Propidium Iodide DNA content analysis of control untreated BGO1V cells (<b>G</b>). Propidium Iodide DNA content analysis of JC011 treated BGO1V after 6 hrs showing a rapid increase in the sub-G1 fraction (<b>H</b>). R1 = sub-G1 fraction, R2 = G1 fraction, R3 = early/late S-phase fraction, R4 = G2/M fraction.</p