Mechanism-based Facilitated Maturation of human pluripotient stem cell-derived Cardiomyocytes

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ABSTRACT

The attrition rates of drugs in development, many of which attributed to unforeseen cardiotoxic side effects of the drugs being tested in humans that were not realized in preclinical animal models, are a significant problem facing the pharmaceutical industry. Recent advances in human stem cell biology have paved the way for incorporating human cell models into the two key aspects of developing new drugs: discovering new effective entities and screening for their safety. Functional cardiomyocytes can now be derived from human pluripotent stem cells (hPSCs), including both embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Moreover, recent studies demonstrate the ability of cardiomyocytes derived from patients' iPSCs to recapitulate the phenotype of several known cardiac diseases. In the present review we describe the knowledge recently gained on this promising human cell source in order to fulfill its potential as a useful tool for drug screening. Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.

Keywords: Human pluripotent stem cells; Cardiomyocytes; Drug screening: Human induced pluripotent stem cell; Human embryonic stem cell; Cardiomyocytes; Drug loading test; and Arrhythmia

ABSTRACT2

CHAPTER 1: INTRODUCTION4

CHAPTER 2: LITERATURE REVIEW6

Cardiotoxicity6

Adverse cardiac effects6

Mechanisms and models7

Cardiomyocytes derived from human pluripotent stem cells8

Cardiac differentiation8

Cardiac marker expression profile9

Structural properties10

Electrophysiological properties10

Ion channel expression11

Excitation-contraction coupling12

Beating rate13

Adrenergic and cholinergic responses13

Pharmacological Responses14

Electrophysiology15

Microelectrode and patch clamp recordings15

Field potentials16

Optical mapping16

Ionic currents17

Cell-cell coupling20

Gap junctions20

CHAPTER 3: DATA AND METHODOLOGY21

Materials and Methods21

Human iPS and human ES cell culture21

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)22

Immunohistochemistry23

Electrophysiological examination24

Drug loading test24

Analysis of beating rate and contractility24

Statistics25

CHAPTER 4: RESULTS AND DISCUSSION26

Time Course Analysis of Gene Expression during Cardiac Differentiation26

Cardiac Differentiation of Human iPS cells via EBs26

Electrical analysis of contractile colonies27

Effects of Drugs on the Beating Frequency of Cardiomyocytes28

The effects of Drugs on the Contractility of Cardiomyocytes derived from Human iPS cells30

Drug testing using Human Pluripotent Stem Cells-derived Cardiomyocytes32

Disease modeling34

Discussion34

CHAPTER 5: CONCLUSIONS38

CHAPTER 1: INTRODUCTION

The average time to develop a new drug is between 10 and 15 years and the associated costs can reach the magnitude of one billion USD (Kola & Landis, 2004). The attrition rate of drugs is staggeringly high: more than 90% of the drugs tested in clinical trials fail to be approved. Compounds fail late in clinical testing or even after approval because of a lack of sufficient efficacy or unanticipated toxicity; ...