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.

Figure 1. Morphology of BGO1V-MEF cultures following treatment with 20 mM JC011 for 12 hrs.

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

Figure 2. Dose response cytotoxicity data.

Dose response curves for 3 PSC cell lines (BGO1V, H9 and iPS-Foreskin-1) following treatment with JC011 (A). Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011 (B). Time course cytotoxicity analysis for differentiating BGO1V cultures treated with JC011 at 4-day intervals following bFGF withdrawal (C). 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 (D). Cell viability values were normalized to untreated controls and reported as mean ± S.D. of three independent experiments (n = 3).

Figure 3. Table of structure, molecular mass, ORAC and DPPH antioxidant data for 6 JC analogues (JC005, JC007, JC010, JC011, JC017 and JC040).

Figure 4. JC011 treated PSCs fail to form teratomas in SCID mice and can be effectively used to enrich for specialized cells.

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 (A–D). 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 (E). 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 (F).

Figure 5. Comparative microarray analysis reveals involvement of the PERK/ATF4/DDIT3 ER stress pathways.

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

Figure 6. JC011 and JC040 reduce ROS levels in PSCs.

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 (A). JC007 (non-cytotoxic) does not alter endogenous ROS levels while JC005 (non-cytotoxic) increases ROS levels but without any corresponding cytotoxicity to BGO1V (B). FACS histograms are representative outcomes of 4 independent experiments. DCHF-DA stained JC011 treated BGO1V cells (C, D). DCHF-DA stained JC005 treated BGO1V cells (E, F).

Figure 7. siRNA knockdown of the key ER stress genes ATF-4 and DDIT3 leads to reduced sensitivity towards JC011 in NCCIT cells.

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 (A–E). qRT-PCR confirmation of ATF-4 and DDIT3 transcript knockdown (F). 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.