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Thyroid hormone and development of cardiomyocytes derived from human embryonic and induced pluripotent stem cells

Final Report Summary - T3D3 STEM CELLS (Thyroid hormone and development of cardiomyocytes derived from human embryonic and induced pluripotent stem cells)

Functional cardiomyocytes can be derived from both embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. In addition to their regenerative potential investigate for tissue repair they are being developed as a model system for basic research or drug discovery/toxicology investigations. They represent an hope in term of progress on cardiovascular disease research since their human origin, long survival in culture and relative easy of transfection give them distinct advantages over the adult and neonatal models currently used. However, from both hESC and hiPSC, the stem cell-derived cardiomyocyte (SC-CM) phenotype is immature, and resemble to foetal or neonatal cardiomyocytes.
In the adult heart thyroid hormone (TH) has pleiotropic effects on contractility, energy metabolism and mitochondrial remodelling. During development, Deiodinase 3 (D3) metabolises TH into inactive products to protect the foetus against high maternal Triiodothyronine (T3) levels. Down regulation of D3 at appropriate times during development allows T3 action on physiological growth and maturation of the heart. The aim of this study is to drive SC-CM to a more mature state by modulation of TH pathway.
hiPSC-CM, (Cellular Dynamics) were treated with 3nM or 30nM of T3 for up to 4 weeks. Gene expression of TH target genes that are key markers of cardiac maturation or oxidative metabolism were assessed by qPCR. The expression of beta 1 Adrenergic Receptor (ADR) was increased in T3 treated cells and remained stable for up to 4 weeks (p<0.05). The alpha/beta myosin heavy chain (MHC) ratio was increased in correlation with T3 concentrations at both 1, 2 and 4 weeks (p<0.001). ATP synthase beta subunit (involved in the electron transport chain) expression level was increased after 2 weeks of treatment with 30nM of T3 (p<0.05). No significant change in gene expression was observed for Sarcoplasmic Reticulum Ca2+-ATPase (SERCA), sodium calcium exchanger (NCX), ryanodine receptor (RYR2), pyruvate dehydrogenase kinase 4, cytochrome c and cytochrome b.
Immunostaining for troponin T and TMRM (mitochondrial membrane potential, dye) was used to measure cell size and mitochondrial density and network organisation respetivly. Nuclei were stained with Hoechst. Plates were scanned on ArrayScan™ VTI automated microscopy and image analysis system (Cellomics). T3 did not increase hiPSC-CM size. No modification of the mitochondrial density or network organisation was observed on treated cells with T3.
The spontaneous beating rate of treated cells (T3, 30nM) was increased 1.95 +/- 0.09 and 2.10 +/- 0.73 time in comparison to control at 2 and 4 weeks respectively (p<0,001). In response to CGP (10M), an inverse agonist of the beta1 ADR, beating rate of T3 treated cells return to a level similar to the control ones. These results correlated beta1 ADR expression and the beating rate modulation.
Direct visualization of intracellular Ca2+ in cardiomyocytes using Fluo-4 AM was used to study Ca2+ dynamics. Ca2+ transient data was acquired using live cell imaging with hiPSC-CM spontaneously beating or under field stimulation (0.7 Hz, 0.8Hz and 1 Hz) using an external pacing generator. At 2 weeks, the spontaneous time to peak, time to 50%, 75% and 90% of relaxation and transient duration were significantly decreased in correlation with T3 concentrations (p<0.05). Under forced pacing by field stimulation the time to peak shortening is confirmed (p<0.05). Whatever the T3 concentration or field stimulation, the amplitude did not change. Despite no change in term of Ca2+ handling molecules expression, functional analysis highlighted Ca2+ dynamics modulation in accordance to a more mature status of the cells.
D3 gene expression was compared by qPCR in hiPSC-CM, hESC (undifferentiated H7 cell line) and hESC-CM, generated 1) via embryoid body (EB) formation, or 2) from dense cell monolayers treated with Activin A and bone morphogenetic protein 4 (Activin A/BMP4). hESC-CM were maintained in culture for up to 6 months. At 1 month post-differentation, D3 expression in the hiPSC-CM was very low in comparison to hESC-CM. Normalized by hESC, D3 expression levels of hiPSC-CM, hESC-CM via Activin A /BMP4 and hESC-CM via EB were 0.02 +/- 0.01 9.38 +/- 0.24 and 2.06 +/- 0.7 fold change respectively (p<0.001). In comparison to hESC, a peak in D3 expression was seen at 1 and 2 months post-differentiation of hESC-CM (9.38 +/- 0.24 and at 8.53 +/- 2.35 time increase respectively, p<0.001) and then decreased.
Immunostaining against troponinT and D3 highlighted the presence of the enzyme in the cells. Simultaneous application of iopanoic Acid (IOP), an inhibitor of the enzyme, and T3 was tested as strategy to drive the maturation of hESC-CM. In comparison to control, treatment with either T3 alone and T3 + IOP induced a significant increase of the alpha/beta myosin heavy chain (MHC) ratio: 4.21 +/- 0.08 and 8.03 +/-4.75 respectively (p<0.05). SERCA level of expression did not change. No modification of phenotypical change (cell size) was observed.

Conclusion: T3 treatment of hiPSC-CM induced modulation of some T3 specific target genes and/or function including key markers of cardiac maturation and oxidative metabolism at different time points. Low D3 expression level in hiPS-CM in comparison to hESC-CM indicated a difference between stem cell sources. Moreover D3 could be an intrinsic control mechanism of hESC-CM maturation. Treatment with high dose of T3 (30nM) and/or IOP could modulate some markers of the cardiac maturation.