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Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies
Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu
Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu
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Resource and Technical Advance Cardiology Stem cells

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies

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Abstract

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Authors

Tony Chour, Lei Tian, Edward Lau, Dilip Thomas, Ilanit Itzhaki, Olfat Malak, Joe Z. Zhang, Xulei Qin, Mirwais Wardak, Yonggang Liu, Mark Chandy, Katelyn E. Black, Maggie P.Y. Lam, Evgenios Neofytou, Joseph C. Wu

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Figure 1

Electrophysiological assessment of ESC-CMs after minimal dose doxorubicin treatment.

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Electrophysiological assessment of ESC-CMs after minimal dose doxorubici...
(A) Representative local extracellular action potential (LEAP) tracings recorded at the multicellular monolayer level under control (top) and doxorubicin treatment (bottom) conditions. (B) Box-and-whisker plots representing beating rate (left) and LEAP potential duration (LPD) at 50% and 90% repolarization (right; analogous to APD) at the multicellular monolayer level. n = 18 per group. (C) Representative fluorescent intensities over transmembrane distance and time and action potential tracings at the single-cell level, as measured by the voltage sensor, ASAP2. (D) Box-and-whisker plots depicting beating rate (left) and APD at 50% and 90% repolarization (right). n = 23 per group. (E) Representative AP tracings recorded from patch clamp of single “ventricular-like” control (top left) and doxorubicin-treated ESC-CMs (bottom left) and “atrial-like” control (top right) and doxorubicin-treated ESC-CMs (bottom right). (F) Scatter plot displaying control and doxorubicin-treated “ventricular-like” (n = 10 for untreated, n = 9 for 0.01 μmol/L doxorubicin) and “atrial-like” ESC-CMs (n = 6 for untreated and 0.01 μmol/L doxorubicin) beating rate (left) and APD (right). Action potential durations at 50% and 90% repolarization (APD50, APD90); and LEAP potential duration at 50% and 90% repolarization (LPD50, LPD90). n = 9 per group. Differences between the untreated group and treatment group were not significant. Statistical analysis was performed with a 2-tailed Student’s t test comparing viability of untreated cells to cells treated with each doxorubicin dose. *P < 0.05, **P < 0.0001. Data represent mean ± SEM.

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