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.
Highlights
Over the last two decades, rapid advances in pluripotent stem cells (PSCs), which comprise embryonic stem cells (ESCs) and induced pluripotent stem cell, have allowed regenerative therapy in clinical trials [1, 2]
Based on fluorescence-activated cell sorting (FACS) analysis of cells stained for cardiac markers troponin T (13-11, Thermo Scientific, MA512960) and α-actinin (H-300, Santa Cruz Biotechnology, sc-15336), we determined that cardiac cell purity was 80%–90%, as is typical with our differentiation protocol [21]
90% cell death was observed in ESCs treated with minimal dose doxorubicin (0.01 μmol/L) for 48 hours compared with untreated ESCs (P < 0.05)
Summary
Over the last two decades, rapid advances in pluripotent stem cells (PSCs), which comprise embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs), have allowed regenerative therapy in clinical trials [1, 2]. Combined with novel methods to efficiently produce PSC-derived cardiomyocytes (CMs), regenerative therapies to treat heart diseases are feasible [3]. Most regenerative therapies targeting cardiovascular disease involve using stem cell–based therapies to replace injured myocardium and repair injured cardiac tissue to restore ventricular function [4]. In the case of cell therapy for patients with heart disease, transplanted cells are intended to engraft onto the endogenous myocardium to restore or enhance cardiac function. The majority of injected cells will neither engraft nor survive following implantation. Injecting a higher cell dosage to compensate can proportionally increase the risk of residual undifferentiated PSCs, and the risk of unintended teratoma formation [6, 7]
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