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Scalable, reproducible, and economical method for producing cardiomyocytes from human iPS cells
196
Abstracts
0129 Application of optical measurements of electrical activity to Cor4U human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) as a predictive tool for preclinical safety assessment Maria P. Hortigon-Vinagrea, Amy E. Taylorb, Silke Schwengbergc,d, Bettina Bertramc, Victor Zamoraa, Ana Costab, Margaret A. Craigb, Francis L. Burtona,b, Godfrey L. Smitha,b a
University of Glasgow, Glasgow, UK Clyde Biosciences, Glasgow, UK c 4 Cells at Work Consulting & Services, Dueren, Germany d Axiogenesis AG, Cologne, Germany b
hiPSC-CMs are a useful predictive tool for cardiac safety assessment. This reproducible and relevant cell model provides a primary like phenotype, including normal electrophysiology and pharmacology. Appropriate action potential characteristics and drug sensitivity are crucial for cardiac toxicity. Using the optical platform CellOPTIQ (Clyde Biosciences, UK), the transmembrane potential of Cor4U hiPSC-CMs (Axiogenesis, Germany) was examined under conditions of spontaneous activity and in the presence of standard drugs. Electrical activity was monitored from spontaneously beating Cor4U hiPSC-CMs (3–7 days in vitro) plated in 96 wells glass bottom plates (20,000 cells/well) using the fluorescence dye di-4-ANEPPS. The records were subsequently analysed off-line using proprietary software (Clyde Biosciences). Baseline parameters (N = 104) were as follows: cycle length, 0.8 s ± 0.02; APD50 and APD90 were 198.5 ms ± 2.3 and 269.4 ms ± 4.7, respectively whereas Trise was 2.9 ms ± 1.65. E4031 (hERG blocker), nifedipine (L-type Ca2 + channel blocker) and flecainide (Na+ channel blocker) were assessed. E4031 caused a dose dependent increase on APD90, 100 nM caused an increase to 148.34 ± 17.8% of baseline, p b 0.005 (n = 9) without any significant effect on cycle length (100.54 ± 9.9% (n = 9, p = 0.8)). Na+ channel blockade by flecainide was evident from a dose dependent increase of TRise, 145 ± 19.5%; 197 ± 29.6% and 232 ± 55.5% of baseline for 0.3, 1 and 3 μM respectively, p b 0.05 (n = 9). L-type Ca-channel blockade by nifedipine caused a significant shortening of APD at 0.1 and 0.3 μM (73.64 ± 5.7 and 58.05 ± 7.6% change of baseline, respectively, p b 0.05 (n = 10)). This suggests that Cor4U hiPSC-CMs have the appropriate basal electrophysiology profile and drug sensitivities to facilitate cardiotoxicology screening. Acknowledgements: Fundacion Alfonso Martin Escudero.
doi:10.1016/j.vascn.2015.08.127
0130 Functional effects of cardiac reference agents in human IPSC cardiomyocytes correlate with gene expression profile Minxue Huang, Wen-Pin Yang, Jun Zhu, Hong Shi, Lewis Buchanan, Jae Kwagh, Paul Levesque Bristol Myers Squibb, Pennington, NJ, USA Human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) are widely used in assessing drug-induced cardiotoxicity. During validation of cardiac functional assays with hIPSCs on various platforms including impedance, multielectrode array electrophysiology and cellular metabolism measurement system, it became apparent that while some reference agents produced anticipated effects, others did not. To understand possible reasons
for lack of translation, gene expression profiling of hIPSC-CMs and human adult heart using Affymetrix chips was conducted. The focus was mostly on cardiac ion channels and chronotropic or inotropic target genes which were the targets of the reference agents used in functional studies. In general, and consistent with literature, ion channels were expressed in hIPSCs as expected, although expression of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) was higher in hIPSC than in human adult heart. Not surprisingly, ion channel reference agents produced anticipated effects in hIPSC across functional platforms. Most cardiac contractility-associated related genes were also expressed in hIPSC CMs, although some had lower or minimal expression such as calsequestrin 2, ryanodine receptor 2 and adrenoceptor beta 1, which were expressed at 20% compared to human adult heart. Type 1 angiotensin II receptor and phosphodiesterase 3A had minimal expression in hIPSCs. The gene expression profiling results were consistent with activity or lack thereof of standard reference agents that modulate cardiac function. The results indicate that hIPSC CMs will have mixed utility for CV discovery or toxicology applications depending on the targets of or mechanism of interest.
doi:10.1016/j.vascn.2015.08.128
0131 Scalable, reproducible, and economical method for producing cardiomyocytes from human iPS cells Masamichi Itoa, Yuki Kuramotob, Atsuhiko Naitoa,b a
The University of Tokyo, Tokyo, Japan Osaka University, Osaka, Japan
b
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are considered to be a promising tool for various purposes, e.g. regenerative medicine, drug discovery, and safety pharmacology. Many researchers have reported various protocols for inducing cardiac differentiation from hiPSC. For the above-mentioned purposes, ideal protocol for cardiac differentiation should be scalable (3D-culture preferred), inexpensive (use of small molecule preferred), and highly reproducible (defined factor preferred). Here, we report an innovative protocol of cardiac differentiation, which is based on widely used protocol from Gordon Keller's lab that uses various cytokines in a 3D-culture system (Yang L et al. Nature 2008). In the new protocol, costs for inducing cardiac differentiation were reduced by 96% with improved differentiation efficiency and high interclonal reproducibility. We identified that small molecule that activates Wnt signaling can replace a cytokine that was used for mesoderm induction (Activin A and BMP) and that inhibitor of Wnt signaling, in combination with inhibitor of Tgfb signaling and a steroid hormone can induce cardiac differentiation more potently than combination of cytokine that was used after mesoderm induction (Dkk-1, bFGF, and VEGF). We have also developed a differentiation medium that consisted of defined and inexpensive factors. The total cost for inducing cardiac differentiation was cut from 1,376,250 yen (~$13,762) to 57,150 yen (~$571) in a 1 L scale. Our new protocol and culture medium would dramatically reduce the cost for producing cardiomyocytes from iPSCs and would promote utilization of iPSC-derived cardiomyocytes for drug discovery and safety pharmacology.
doi:10.1016/j.vascn.2015.08.129
Abstracts
0129 Application of optical measurements of electrical activity to Cor4U human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) as a predictive tool for preclinical safety assessment Maria P. Hortigon-Vinagrea, Amy E. Taylorb, Silke Schwengbergc,d, Bettina Bertramc, Victor Zamoraa, Ana Costab, Margaret A. Craigb, Francis L. Burtona,b, Godfrey L. Smitha,b a
University of Glasgow, Glasgow, UK Clyde Biosciences, Glasgow, UK c 4 Cells at Work Consulting & Services, Dueren, Germany d Axiogenesis AG, Cologne, Germany b
hiPSC-CMs are a useful predictive tool for cardiac safety assessment. This reproducible and relevant cell model provides a primary like phenotype, including normal electrophysiology and pharmacology. Appropriate action potential characteristics and drug sensitivity are crucial for cardiac toxicity. Using the optical platform CellOPTIQ (Clyde Biosciences, UK), the transmembrane potential of Cor4U hiPSC-CMs (Axiogenesis, Germany) was examined under conditions of spontaneous activity and in the presence of standard drugs. Electrical activity was monitored from spontaneously beating Cor4U hiPSC-CMs (3–7 days in vitro) plated in 96 wells glass bottom plates (20,000 cells/well) using the fluorescence dye di-4-ANEPPS. The records were subsequently analysed off-line using proprietary software (Clyde Biosciences). Baseline parameters (N = 104) were as follows: cycle length, 0.8 s ± 0.02; APD50 and APD90 were 198.5 ms ± 2.3 and 269.4 ms ± 4.7, respectively whereas Trise was 2.9 ms ± 1.65. E4031 (hERG blocker), nifedipine (L-type Ca2 + channel blocker) and flecainide (Na+ channel blocker) were assessed. E4031 caused a dose dependent increase on APD90, 100 nM caused an increase to 148.34 ± 17.8% of baseline, p b 0.005 (n = 9) without any significant effect on cycle length (100.54 ± 9.9% (n = 9, p = 0.8)). Na+ channel blockade by flecainide was evident from a dose dependent increase of TRise, 145 ± 19.5%; 197 ± 29.6% and 232 ± 55.5% of baseline for 0.3, 1 and 3 μM respectively, p b 0.05 (n = 9). L-type Ca-channel blockade by nifedipine caused a significant shortening of APD at 0.1 and 0.3 μM (73.64 ± 5.7 and 58.05 ± 7.6% change of baseline, respectively, p b 0.05 (n = 10)). This suggests that Cor4U hiPSC-CMs have the appropriate basal electrophysiology profile and drug sensitivities to facilitate cardiotoxicology screening. Acknowledgements: Fundacion Alfonso Martin Escudero.
doi:10.1016/j.vascn.2015.08.127
0130 Functional effects of cardiac reference agents in human IPSC cardiomyocytes correlate with gene expression profile Minxue Huang, Wen-Pin Yang, Jun Zhu, Hong Shi, Lewis Buchanan, Jae Kwagh, Paul Levesque Bristol Myers Squibb, Pennington, NJ, USA Human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) are widely used in assessing drug-induced cardiotoxicity. During validation of cardiac functional assays with hIPSCs on various platforms including impedance, multielectrode array electrophysiology and cellular metabolism measurement system, it became apparent that while some reference agents produced anticipated effects, others did not. To understand possible reasons
for lack of translation, gene expression profiling of hIPSC-CMs and human adult heart using Affymetrix chips was conducted. The focus was mostly on cardiac ion channels and chronotropic or inotropic target genes which were the targets of the reference agents used in functional studies. In general, and consistent with literature, ion channels were expressed in hIPSCs as expected, although expression of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) was higher in hIPSC than in human adult heart. Not surprisingly, ion channel reference agents produced anticipated effects in hIPSC across functional platforms. Most cardiac contractility-associated related genes were also expressed in hIPSC CMs, although some had lower or minimal expression such as calsequestrin 2, ryanodine receptor 2 and adrenoceptor beta 1, which were expressed at 20% compared to human adult heart. Type 1 angiotensin II receptor and phosphodiesterase 3A had minimal expression in hIPSCs. The gene expression profiling results were consistent with activity or lack thereof of standard reference agents that modulate cardiac function. The results indicate that hIPSC CMs will have mixed utility for CV discovery or toxicology applications depending on the targets of or mechanism of interest.
doi:10.1016/j.vascn.2015.08.128
0131 Scalable, reproducible, and economical method for producing cardiomyocytes from human iPS cells Masamichi Itoa, Yuki Kuramotob, Atsuhiko Naitoa,b a
The University of Tokyo, Tokyo, Japan Osaka University, Osaka, Japan
b
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are considered to be a promising tool for various purposes, e.g. regenerative medicine, drug discovery, and safety pharmacology. Many researchers have reported various protocols for inducing cardiac differentiation from hiPSC. For the above-mentioned purposes, ideal protocol for cardiac differentiation should be scalable (3D-culture preferred), inexpensive (use of small molecule preferred), and highly reproducible (defined factor preferred). Here, we report an innovative protocol of cardiac differentiation, which is based on widely used protocol from Gordon Keller's lab that uses various cytokines in a 3D-culture system (Yang L et al. Nature 2008). In the new protocol, costs for inducing cardiac differentiation were reduced by 96% with improved differentiation efficiency and high interclonal reproducibility. We identified that small molecule that activates Wnt signaling can replace a cytokine that was used for mesoderm induction (Activin A and BMP) and that inhibitor of Wnt signaling, in combination with inhibitor of Tgfb signaling and a steroid hormone can induce cardiac differentiation more potently than combination of cytokine that was used after mesoderm induction (Dkk-1, bFGF, and VEGF). We have also developed a differentiation medium that consisted of defined and inexpensive factors. The total cost for inducing cardiac differentiation was cut from 1,376,250 yen (~$13,762) to 57,150 yen (~$571) in a 1 L scale. Our new protocol and culture medium would dramatically reduce the cost for producing cardiomyocytes from iPSCs and would promote utilization of iPSC-derived cardiomyocytes for drug discovery and safety pharmacology.
doi:10.1016/j.vascn.2015.08.129