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. 2021 Jul 29;22(15):8114.
doi: 10.3390/ijms22158114.

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity

Lauren Michelle Walker  1 , Nicole R L Sparks  1 , Veronica Puig-Sanvicens  1 , Beatriz Rodrigues  1 , Nicole I Zur Nieden  1
Affiliations

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity

Lauren Michelle Walker et al. Int J Mol Sci. .

Abstract

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.

Keywords: cardiomyocytes; embryonic stem cell test; embryotoxicity screen; induced pluripotent stem cells; tobacco smoke solution.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
hiPSCs consistently and robustly differentiated into cardiomyocytes. Differentiated cardiomyocytes were assessed for cardiomyocyte-specific markers and gene expression. (A) Schematic protocol for cardiomyocyte differentiation and embryotoxicity screen. (B) Immunocytochemistry stains of differentiated cultures confirmed cardiomyocyte identity via myosin heavy chain (MHC) and Troponin I (Trop I). (C) Differentiated cardiomyocytes expressed cardiac-specific genes TBX5 and MEF2c as measured by qPCR, n = 3 biological replicates ± SD, * p < 0.05, Student’s t-test. hiPSC, human induced pluripotent stem cell; MHC, myosin heavy chain; Trop I, Troponin I.
Figure 2
Figure 2
Presence of contractile cardiomyocyte clusters declines with treatment with embryotoxicants 5-FU and atRA. hiPSCs were treated with different concentrations of 5-FU, atRA, or PenG and photographed. hiPSC, human induced pluripotent stem cell; 5-FU, 5-fluorouracil; atRA, all-trans retinoic acid; PenG, penicillin G.
Figure 3
Figure 3
Treatment with embryotoxicants 5-FU and atRA impeded cardiomyocyte differentiation. hiPSCs were treated with different concentrations of 5-FU, atRA, or PenG and evaluated for differentiation inhibition by visually scoring the number of actively contracting cardiomyocyte clusters. Each data point represents the mean of three independent experiments ± SD. *: p < 0.05 = the lowest concentration significantly below the untreated control group as determined by One-Way ANOVA. hiPSC, human induced pluripotent stem cell; 5-FU, 5-fluorouracil; atRA, all-trans retinoic acid; PenG, penicillin G.
Figure 4
Figure 4
5-FU and atRA treatment reduced hiPSC-cardiomyocyte and hFF viability in a dose-dependent manner as assessed via MTT assay. (A) hiPSC viability screens for 5-FU, atRA, and PenG, n = 3 biological replicates ± SD. *: p < 0.05 = the lowest concentration significantly below the untreated hiPSC control group as determined by One-Way ANOVA. (B) hFF viability screens for 5-FU, atRA, and PenG, n = 3 biological replicates ± SD. *: p < 0.05 = the lowest concentration significantly below the untreated hFF control group as determined by One-Way ANOVA. hiPSC, human induced pluripotent stem cell; MTT, mitochondrial dehydrogenase activity assay; 5-FU, 5-fluorouracil; atRA, all-trans retinoic acid; PenG, penicillin G; hFF, human foreskin fibroblast.
Figure 5
Figure 5
Treatment with embryotoxicants 5-FU and atRA impeded cardiomyocyte differentiation as measured by day 10 TBX5 gene expression in hiPSCs. hiPSCs were treated with different concentrations of 5-FU, atRA, or PenG and evaluated for TBX5 or MEF2c expression via qPCR. Data points represent means of three independent experiments ± SD. Inhibition of differentiation (ID50) was determined from dose–response curves as a 50% reduction in gene expression in the control. hiPSC, human induced pluripotent stem cell; 5-FU, 5-fluorouracil; atRA, all-trans retinoic acid; PenG, penicillin G. *: p < 0.05 = the lowest concentration significantly below the untreated hiPSC control group as determined by One-Way ANOVA.
Figure 6
Figure 6
Effects of cigarette smoke and Snus smokeless tobacco on developing cardiomyocytes. (A) Contractile and viability screens for hiPSCs exposed to MS cigarette smoke using contractile and MTT assay, n = 3 ± SD. (B) Contractile and viability screens for hiPSCs exposed to Snus smokeless tobacco using contractile and MTT assay, n = 3 ± SD. (C) Viability screen for hFFs exposed to MS cigarette smoke using MTT assay, n = 3 ± SD. (D) Viability screen for hFFs exposed to Snus smokeless tobacco using MTT assay, n = 3 ± SD. hiPSC, human induced pluripotent stem cell; MTT, mitochondrial dehydrogenase activity assay; MS; mainstream; hFF, human foreskin fibroblast. *: p < 0.05 = the lowest concentration significantly below the untreated hiPSC control group as determined by One-Way ANOVA.
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References

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