hamaguchi ryoko biothesis - stanford university · school application process. i would also like to...
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HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES AS AN IN VITRO MODEL OF DOXORUBICIN-INDUCED CARDIOTOXICITY
An Honors Thesis Submitted to
The Department of Biology In partial fulfillment of the Honors Program
STANFORD UNIVERSITY
By RYOKO HAMAGUCHI
MAY 07, 2015
20 May 2015
PREFACE
This thesis is original, unpublished, independent work by the author, R. Hamaguchi.
ACKNOWLEDGEMENTS
First and foremost, I would like to thank Dr. Sean M. Wu for his guidance since I have
joined his lab in the spring of my sophomore year. By encouraging my independence in
designing and executing this thesis, he has allowed me to fully experience and appreciate the
challenges and joys of scientific research. He has not only been an incredibly supportive research
advisor, but also an indispensable source of advice and encouragement throughout my medical
school application process. I would also like to thank my primary lab supervisor, Arun Sharma,
for his unparalleled mentorship and friendship. His instruction and support have allowed me to
achieve a truly meaningful undergraduate research experience that will be a foundation for my
future scholarly work in medical school and beyond. While I entered the lab with minimal
exposure to cell culture and other wet lab skills, Arun has helped me to not only gain experience
in these techniques, but also accumulate confidence in the full arc of research—from the
conceptualization of a project to the execution of experiments and the analysis of data. I cannot
thank him enough for giving me the strength to push through the disappointing failures and
celebrating with me in my moments of triumph.
I would also like to express my gratitude for all current and past members of the Sean
Wu Lab, whose support and encouragement has been paramount to my growth as a student and
undergraduate researcher. I would also like to thank the members of the Joseph C. Wu Lab,
including Dr. Ioannis Karakikes, who guided me in designing and optimizing the RPA-based
iron fluorescence assays and provided many of the necessary compounds for the experiments. I
am especially grateful for Dr. Paul Burridge, who kindly provided the DOX CONTROL and
DOX TOX human induced pluripotent stem cell lines and whose extensive experience on
doxorubicin’s effects on hiPSC-CMs inspired this thesis work. I have enormous respect for his
genuine investment in mentorship, unrelenting perseverance and sheer brilliance as a scientist. It
has been an absolute privilege to be mentored by him, and I wish him the very best of luck and
continued success as he goes on to lead his new laboratory at the Feinberg School of Medicine at
Northwestern University.
In addition to the labs in which I conducted my research, this thesis would not be possible
without the support from the larger Stanford community and its many academic departments. I
would like to thank Dr. Scott J. Dixon (Dept. of Biology) for his support and thoughtful
feedback as the second reader of my thesis. I would like to thank the Department of Biology at
Stanford University, including my faculty advisor, Dr. Patricia Jones, and the coordinators of
the Honors in Biology program, including Dr. Jamie Imam and Ms. Carolynn Beer. I would
also like to thank Dr. Russ Carpenter, whose winter quarter Writing in the Major course,
BIO199W, served as an invaluable resource as I began my thesis-writing process. I would also
like to thank Stanford Bio-X and its Undergraduate Summer Research Program, which helped to
fund my preliminary research for this thesis project.
Finally, and most importantly, I would like to thank my mother and father, Junko and
Hideshi, and my younger brother, Shunsei, for their unending faith in my ambitions. My family
is my most important foundation and source of motivation, and this honors thesis is equally as
much a testament to their support and encouragement as it is a culmination of my own efforts.
TABLE OF CONTENTS LIST OF TABLES ………………………………………………………………………...1
LIST OF FIGURES ……………………………………………………………………….2
ABSTRACT ………………………………………………………………………………..4
INTRODUCTION ………………………………………………………………………... 6
MATERIALS AND METHODS ………………………………………………………... 12
RESULTS ………………………………………………………………………………… 20
DISCUSSION …………………………………………………………………………….. 26
REFERENCES …………………………………………………………………………... 37
TABLES ………………………………………………………………………………….. 44
FIGURE LEGENDS …………………………………………………………………….. 46
FIGURES ………………………………………………………………………………… 51
1
LIST OF TABLES
Table 1. Cell lines and reprogramming methods
Table 2. Primary antibodies used for immunofluorescence staining
Table 3. Summary of Taqman probes used for qPCR assays
2
LIST OF FIGURES
Fig 1. Schematic demonstrating the generation of hiPSC-CMs from patient-specific hiPSCs.
Fig 2. hiPSC-CMs demonstrate dose-dependent cytotoxicity in response to in vitro DOX
treatment.
Fig 3. Transcriptional analysis of ABCB8 expression in DOX-treated hiPSC-CMs.
Fig 4. Patient-specific hiPSC-CMs exhibit variable baseline expression levels of ABCB8, ACO1
and TOP2B.
Fig 5. Application of iron fluorescent sensor to quantify mitochondrial iron accumulation in
DOX-treated hiPSC-CMs.
Supplementary Fig 1. hiPSCs express key pluripotency markers and hiPSC-CMs express
appropriate intracellular sarcomeric proteins.
Supplementary Fig 2. Schematic of iron metabolism genes and their gene products.
Supplementary Fig 3. hiPSC-CMs demonstrate dose-dependent reduction in cell viability and
mitochondrial structural integrity in response to iron pre-treatment.
3
Supplementary Fig 4. Two proposed models for the biological and molecular mechanisms
underlying DIC.
4
ABSTRACT
Doxorubicin (DOX; trade name Adriamycin) is an anthracycline antibiotic and widely
used chemotherapeutic agent. Despite its powerful and wide-spectrum anticancer effects, its
clinical utility is limited by its cardiotoxicity, which increases the risk of dilated cardiomyopathy
and subsequent heart failure among DOX-treated cancer patients and survivors. Recent animal-
based studies have suggested that DOX-induced cardiotoxicity (DIC) is due, at least in part, to
iron accumulation and resulting oxidative stress, and that this is mediated by the downregulation
of ABCB8, which encodes a mitochondrial iron transporter protein called ATP-binding cassette
subfamily B member 8 (ABCB8). Circumventing the challenges of validating these findings in
primary human heart tissue, this study applies human iPSC-derived cardiomyocytes (hiPSC-
CMs) as a surrogate human platform to probe the pathogenic processes of DIC. I hypothesized
that DOX causes dose-dependent ABCB8 downregulation and mitochondrial iron accumulation
in hiPSC-CMs, consistent with studies performed on animal-based in vitro models. Patient-
specific hiPSC-CMs, generated from DOX-treated breast cancer patients who experienced
cardiotoxicity (DOX TOX) and those who did not (DOX CONTROL), were utilized to
investigate whether patient-specific differences in the pattern and severity of ABCB8 expression
and mitochondrial accumulation of iron could predict susceptibility to DIC. I found that, while
high concentrations of DOX caused significant reduction of ABCB8 in hiPSC-CMs, modest and
non-cytotoxic concentrations did not. Further, while no robust difference was observed between
the baseline expression of ABCB8 in DOX CONTROL and DOX TOX cells, these two cancer
patient-derived hiPSC-CM populations both expressed significantly lower levels of ABCB8 than
their HEALTHY CONTROL counterparts. Finally, preliminary data suggested that DOX does
not induce consistent dose-dependent trends in mitochondrial iron accumulation in hiPSC-CMs.
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As the first humanized model of DIC in the specific context of iron metabolism, this work
represents a key step toward improving the clinical utility of DOX, by elucidating the molecular
basis of both its cardiotoxic effects and patient-specific susceptibility to this condition.
(Word count = 312)
Key Words: Induced pluripotent stem cell, cardiomyocyte, cardiotoxicity, iron, doxorubicin
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INTRODUCTION
Clinical context of doxorubicin-induced cardiotoxicity (DIC)
While the development of chemotherapeutic drugs has made incredible contributions
toward improving cancer survival rates, treatment-related morbidities and mortalities represent
significant challenges to physicians and patients1,2,3,4. One key example is doxorubicin (DOX;
trade name Adriamycin), an anthracycline antibiotic widely used as a chemotherapeutic agent for
various adult and pediatric cancers5. DOX exerts its anticancer effects by preventing DNA
replication in proliferative tumor cells through the specific inhibition of topoisomerase 2α
(TOP2A), an enzyme that induces double-stranded DNA breaks, relieving torsional stress and
tangles in coiled DNA6. Today, over three decades since its discovery, DOX remains one of the
most extensively used chemotherapeutic agents in treating malignancies including breast cancer,
lymphoma and sarcoma7. However, its clinical utility is severely hampered by its cardiotoxic
side effects8, which increase the risk of severe dilated cardiomyopathy (DCM) and life-
threatening congestive heart failure among patients who receive DOX as part of their
chemotherapy regimen9. DCM is a progressive disease of the heart muscle, involving the
weakening of the left ventricle and subsequent reduction in the heart’s overall pumping
efficiency10. Congestive heart failure, which is the clinical manifestation of patients with DCM,
is a complex clinical syndrome, resulting from various cardiac disorders (functional or structural)
that hamper the ventricle’s ability to fill with or eject blood11. Given the late onset of these
symptoms, doxorubicin-induced cardiotoxicity (DIC) is a particularly devastating problem
among pediatric cancer patients—of whom approximately 60% are treated with DOX and 10%
of this subpopulation will develop symptomatic cardiomyopathy up to 15 years after treatment
completion5. There is currently no pharmacological cure for DOX-related cardiomyopathy, thus
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leaving many patients with end-stage heart failure with heart transplantation as their sole
therapeutic option (for which they are poor candidates due to their history of cancer)7.
Furthermore, there are dramatic variations in patient-specific sensitivity to DIC—indeed,
histopathologic alterations consistent with cardiotoxicity have been observed at doses as low as
183 mg/m2 (less than a third of the maximum cumulative dose), while other patients have
tolerated doses as high as >1000 mg/m2 with no cardiotoxic side effects12. Given the lack of
robust biomarkers or clinical tests to predict DOX-related cardiomyopathy, prophylaxis and
treatment are often limited to close monitoring for cardiac dysfunction during treatment and
identification of cardiovascular risk factors that may inform the administered dose12.
Research landscape surrounding the mechanisms of DIC
While the underlying pathogenic mechanisms of DIC remain an active locus of research,
excess production of free radicals and associated oxidative stress are thought to be a key
mediator in this multifactorial phenomenon13,14,15. Anthracyclines such as DOX can promote the
formation of reactive oxygen species (ROS) through either the redox cycling of their aglycones
or through complexes formed with iron7. This hypothesis is supported by multiple pieces of
evidence, including the cardioprotective effects of free radical scavengers such as
alphatocopherol16, as well as the reduction in cardiotoxicity observed in transgenic animals in
which endogenous antioxidants such as catalase17, mitochondrial superoxide dismutase18,
thioredoxin-119 and metalothionein20 are overexpressed. The dominant process by which DOX
causes oxidative stress, however, remains unclear. One theory attributes DOX’s cardiotoxicity
to the drug’s off-target inhibitory effects on topoisomerase 2β (TOP2B), an analog of TOP2A
(DOX’s primary anticancer target) that is highly expressed in cardiomyocytes and other
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quiescent, non-proliferative cells8,21,22. Inhibition of this enzyme can cause misregulation of
antioxidant gene expression, mitochondrial biogenesis, and other processes critical to cell
function and survival—many of which can indirectly cause oxidative stress8,21,22.
An alternative theory to the TOP2B hypothesis suggests that DOX directly impairs
processes involved in iron metabolism, causing an intracellular build-up of free labile iron that
amplifies the production of reactive oxygen species (ROS), or free radicals that cause and
exacerbate oxidative stress7. The involvement of iron in DIC is supported by the reported
cardioprotective effects of dexrazoxane (DXZ), an iron chelator, as well as increased
susceptibility to DIC observed in animal models that are overloaded with dietary iron23,24.
Furthermore, recent animal-based studies have suggested that DOX impairs cardiomyocytes’
expression of genes involved in iron metabolism, causing toxic accumulation of iron in the
mitochondria. Recently, Ichikawa et al. used isolated neonatal rat cardiomyocytes (NRCMs) and
in vivo murine DIC models to demonstrate that DOX specifically downregulates the expression
of ABCB825,26. ABCB8 encodes a transporter protein called ATP-binding cassette subfamily B
member 8 (ABCB8), which localizes to the inner mitochondrial membrane and has been shown
to facilitate the export of iron out of the mitochondria25,26. Through a series of in vitro
experiments, the researchers demonstrated that DOX causes downregulation of ABCB8 and
mitochondrial iron accumulation in NRCMs26. Further, knockdown and overexpression of the
gene were shown to exacerbate and reduce mitochondrial iron accumulation and ROS levels,
respectively26. These findings indicate that disruption of iron metabolism—specifically impaired
mobilization of iron out of the mitochondria—may serve as the primary underlying mechanism
of DOX-mediated oxidative stress.
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Application of human induced pluripotent stem cell-induced-cardiomyocytes (hiPSC-CMs) as
models of cardiovascular disease
While in vitro and in vivo animal-based models have proved useful in exploring the
mechanisms of DIC, these approaches are inherently limited by interspecies variations in
anthracycline metabolism and susceptibility to DOX-related oxidative stress27, as well as
differences in general cardiac electrophysiology and ion channel expression28. Given these
species-specific variations in cardiac physiology, animal-based models are not always predictive
of drug activity and off-target side effects in human patients. Physiologically relevant human
cardiac models, in turn, are limited because procuring primary cardiomyocytes requires highly
invasive biopsies, and furthermore, these cells are difficult to dissociate and maintain in culture,
rendering them unsustainable for long-term research purposes29. Moreover, given that a vast
majority of cancer patients are treated with multi-drug regimens, this complex clinical picture
makes it exceedingly difficult to parse out the potential confounding factors and use patient
cardiomyocytes to glean meaningful information that specifically pertains to the effects of DOX.
However, with the advent of human induced pluripotent stem cells (hiPSCs) and rapid
improvement in the efficiency of subsequent differentiation protocols, we are now able to
generate humanized in vitro platforms to model and probe the mechanisms underlying various
diseases29 (Fig 1C). As demonstrated by the work of Yamanaka, et al. and subsequent studies,
hiPSCs can be generated by reprogramming adult somatic cells (such as dermal fibroblasts and
peripheral blood mononuclear cells) into an embryonic stem cell-like pluripotent state through
viral-mediated delivery of genetic factors such as OCT3/4, SOX2, KLF4, and MYC30,31,32. These
hiPSCs can be differentiated into various somatic cell types, including functional, beating
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cardiomyocytes—which have since been used as in vitro models of cardiac pathologies such as
long QT syndrome33 and Coxsackievirus-induced myocarditis34.
Objectives and specific aims of thesis
In this thesis project, I tested the hypothesis that hiPSC-CMs can be utilized as a patient-
specific in vitro cardiac model of the iron metabolism alterations implicated in DIC (Fig 1A). In
addition to hiPSC-CMs generated from healthy control patients (HEALTHY CONTROL hiPSC-
CMs), I utilized hiPSC-CMs generated from DOX-treated breast cancer patients who suffered
from post-treatment cardiotoxicity and those who did not. These two sets of patient-specific lines
were referred to as DOX TOX and DOX CONTROL, respectively. Previous findings indicate
that DOX causes greater toxicity in DOX TOX hiPSC-CMs than compared to DOX CONTROL
counterparts, suggesting that these cells may be a useful model to study the mechanisms
underlying patient-specific susceptibility to DIC (Burridge, et al, unpublished data). Encouraged
by these findings, I used the above three cell populations to (1) validate whether DOX causes
ABCB8 downregulation and iron accumulation in hiPSC-CMs and (2) if so, whether the pattern
or severity of either of these phenotypes can inform and predict patient-specific susceptibility to
DIC. After confirming the susceptibility of hiPSC-CMs to dose-dependent cytotoxicity in
response to in vitro DOX treatment, I used real-time quantitative PCR (RT-qPCR) to validate
whether DOX treatment downregulates ABCB8 in a dose-dependent manner, and whether the
patterns of ABCB8 expression were consistent with the clinical status of the patient population
from which the cells were generated from. For example, do DOX TOX hiPSC-CMs exhibit a
more severe downregulation of ABCB8 in response to DOX? Further, do DOX TOX hiPSC-CMs
express lower levels of ABCB8 at a baseline state, even prior to treatment with DOX? Finally, I
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employed a fluorescent reporter to indirectly quantify the intra-mitochondrial iron content of
DOX-treated hiPSC-CMs and compare the degree of mitochondrial iron accumulation among the
three patient-specific cell populations.
As the first patient-specific humanized model of DIC in the specific context of iron
metabolism, these hiPSC-CMs have enabled us to better understand the pathogenic mechanisms
underlying this clinical problem and the variable susceptibility and resistance to this condition. In
the broader context of clinical translation, this hiPSC-CM model provides a promising tool in
expediting the drug discovery process, including the development of cardioprotectants to treat
DIC, less cardiotoxic analogs of DOX, and innovative drug delivery methods to minimize its
toxicity.
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MATERIALS AND METHODS
1. Assessment of DOX-induced cytotoxicity in hiPSC-CM model
1A. Human induced pluripotent stem cells (hiPSCs) and reprogramming methods:
Peripheral blood mononuclear cells were obtained via blood draws from three healthy
control individuals, and dermal fibroblast samples were obtained via skin biopsy from eight
breast cancer patients who had been treated at Stanford University Hospital with 240 mg/m2
DOX or equivalent. Samples were reprogrammed into hiPSCs using Sendai viruses (Life
Technologies) expressing the four Yamanaka factors, or OSKM (OCT3/4, SOX2, KLF4, and
MYC), and were characterized as previously described (Table 1)32. This Sendai virus-based
method achieves reprogramming without foreign gene insertions into the host genome, making it
a practical solution for the generation of transgene-free hiPSCs35. The breast cancer patient
hiPSCs were referred to as DOX CONTROL and DOX TOX lines, and this classification was
determined based on left ventricular ejection fraction (LVEF), which refers to the amount or
percentage of blood that is pumped out of the left ventricle with each heartbeat. DOX TOX
hiPSCs were generated from four patients who experienced cardiotoxicity following their
chemotherapy regimen (as documented by post-treatment LVEF=10-45%), and DOX
CONTROL hiPSCs from four patients who did not (LVEF>56%, Burridge, et al, unpublished
data). Following reprogramming and characterization, hiPSC colonies were cultured on feeder-
free, growth factor-reduced Matrigel (Corning)-coated 6-well tissue culture dishes (Greiner) with
E8 pluripotent stem cell growth medium (Life Technologies). Successful reprogramming was
confirmed by immunofluorescence staining for established pluripotency factors, including
Nanog, Sox2, Oct3/4 and TRA-1-81 (Table 2).
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1B. Cardiac differentiation and generation of hiPSC-derived cardiomyocytes (hiPSC-CMs)
All hiPSC lines (HEALTHY CONTROL, DOX CONTROL and DOX TOX) were
differentiated into beating cardiomyocytes using a previously published 2D monolayer
differentiation protocol and maintained in a 5% CO2/air environment36 (Fig 1B). Briefly, this
protocol employs a chemically defined medium (CDM3), consisting of basal medium RPMI-
1640 (Corning), L-ascorbic acid 2-phosphate (Wako) and rice-derived human recombinant
albumin (Sciencell Research), and reproducibly generates contractile sheets consisting of up to
95% TNNT2 (Troponin T)-positive hiPSC-CMs, with a yield reaching 100 cardiomyocytes for
each input hiPSC36. Unlike earlier protocols that require supplementation of various components
(some of them animal-derived), this new method allows highly efficient generation of hiPSC-
CMs without the use of compounds that may influence the reproducibility of differentiation, as
well as maturation and subtype specification of the resulting cell population36. Once the wells
became approximately 60-70% confluent, 0.5 mM EDTA was used to dissociate hiPSC colonies
into single-cell suspension in E8 media containing 10 μM Rho-associated protein kinase
inhibitor (Biorbyt), which has been shown to improve the survival of human embryonic stem
cells37. Approximately 100,000 cells were replated into 6-well dishes pre-coated in Matrigel.
Once approximately 85% confluent, cells were treated for 2 days (D0-D2, where D0 = start of
cardiac differentiation) with 6 μM CHIR99021 (Selleck Chemicals) in CDM3 to activate Wnt
signaling and induce mesodermal differentiation. Following CHIR treatment, cells were treated
for 2 days (D2-4) with 6 μM Wnt-C59 (Biorbyt) in CDM3 to inhibit Wnt signaling and induce
cardiogenesis. After an additional 4 days on regular CDM3 medium (D4-8), cells were glucose-
deprived for 3-4 days on CDM3 glucose (-) medium for the purpose of hiPSC-CM purification
and elimination of non-cardiomyocyte cells, including smooth muscle and endothelial cells38.
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This is possible because cardiomyocytes harbor the unique metabolic ability to selectively utilize
fatty acids as a source of energy38. Finally, cells were cultured on regular CDM3 medium,
harvested and/or replated for experimental use. When replating, hiPSC-CMs were dissociated
with TrypLE™ Express (Life Technologies) into single cell suspension in re-plating medium,
comprised of CDM3 + 10% fetal bovine serum (FBS) + 10 μM Rho-associated protein kinase
inhibitor, which has been shown to enhance the survival of hiPSC-CMs39, and seeded on
Matrigel-coated plates.
1C. Doxorubicin treatment
Following differentiation and purification (as described above), hiPSC-CMs (D14-20)
were treated with varying concentrations of doxorubicin (DOX) as an in vitro model of the
human heart in a patient undergoing a DOX chemotherapy regimen. 1 mL stock aliquots of 10
mM DOX (in H2O) were prepared and stored at -20°C. The terminal plasma half-life of DOX has
been reported at 20-48 hours, with peak plasma concentration reaching 4 μg/mL (6.9 μM)40.
Thus, hiPSC-CMs were treated with 0, 0.1, 1 and 10 μM DOX (in regular CDM3 medium) for
24 hours, unless stated otherwise.
1D. Cardiac-specific immunofluorescence and visualization of mitochondria
Following dissociation (as described in 1B), hiPSC-CMs were plated on Matrigel-coated
Nunc™ Lab-Tek™ II Chamber Slides (Thermo Fisher Scientific) and treated with 0, 1, 5 and 25
μM DOX for 24 hours. Immunostaining was performed according to previously established
protocols41. Briefly, the cells were washed with Dulbecco’s phosphate buffered saline (DPBS,
Life Technologies), fixed using 4% paraformaldehyde (Electron Microscopy Sciences) and
15
permeabilized with 0.2% Triton™ X-100 (Sigma-Aldrich). Following blocking with 3% bovine
serum albumin (BSA, Sigma-Aldrich), primary antibodies (1:200 dilution) were applied
overnight and secondary antibodies (1:500 dilution) were applied for at least an hour in a dark
environment. Mouse α-actinin (A7811, Sigma-Aldrich) and Goat anti-mouse AlexaFluor® 488
(A11029, Life Technologies) were used as primary and secondary antibodies, respectively
(Table 2). A nuclear co-stain was also performed using the NucBlue® Fixed Cell Stain
ReadyProbes™ reagent (Life Technologies). Slides were covered with Fisherfinest® Premium
Cover Glass slips (Fisher Scientific) prior to visualization. Imaging was conducted on a Zeiss
LSM 510Meta confocal microscope (Carl Zeiss) and analyzed using ZEN imaging software,
both provided at the courtesy of Dr. Kristy Red-Horse (Stanford University, Department of
Biology).
Mitotracker® Green FM (Life Technologies), a green-fluorescent mitochondrial probe,
was utilized for live (real-time) visualization of the mitochondrial morphology in DOX-treated
hiPSC-CMs. Mitotracker® reagents, which contain a mildly thiol-reactive chloromethyl moiety
for labeling mitochondria, passively diffuse across the plasma membrane and accumulate within
active mitochondria. Mitochondrial structures were visualized using a Leica DFC500 fluorescent
microscope and Leica LAS X imaging software, both of which were provided at the courtesy of
Dr. Joseph C. Wu (Stanford Cardiovascular Institute) and his lab.
1E. Quantification of cell viability and DOX-induced apoptosis
The resazurin-based PrestoBlue® Cell Viability Reagent (Life Technologies) was used to
quantitatively assess changes in cell viability in DOX-treated hiPSC-CMs, following the
manufacturer’s protocol. When added to viable, metabolically active cells, the cell-permeant
16
compound in PrestoBlue® is transformed from a blue-colored, non-fluorescent form to a red-
colored, highly fluorescent form (ex: 560nm, em: 590 nm). After 30 minutes of incubation, a
microplate reader (GloMax, Promega) was used to quantify fluorescence levels, which directly
correlate with cell viability and reducing capacity of the intracellular environment.
The JC-10 Microplate Assay Kit (Abcam) was used to quantify the mitochondrial
membrane potential in DOX-treated hiPSC-CMs. Briefly, the JC-10 dye is able to selectively
enter the mitochondria and reversibly change its color from green to orange with increasing
membrane potentials. When excited at 490 nm, membrane polarization causes monomeric JC-10
(em: 520 nm) to aggregate and shift to emission at 570 nm. Thus, relative mitochondrial
membrane potential was quantified as a relative ratio of orange/green fluorescence levels, where
a decrease in ratio was interpreted as a decline in mitochondrial membrane potential, an early
indicator of cell apoptosis.
2. Gene expression analysis
2A. RNA extraction and generation of cDNA
Real-time quantitative PCR (RT-qPCR) was used to asses the expression levels of
ABCB8 and other iron metabolism genes in DOX-treated as well as untreated hiPSC-CMs.
Harvesting of cell samples and RNA extraction was performed using the RNeasy® Mini kit
(Qiagen), following the manufacturer’s protocol. When extracting RNA from small cell samples,
the RNeasy® Micro Kit (Qiagen), which follows a protocol very similar to that of the RNeasy®
Mini Kit, was used to maximize the yield of RNA extracted from each cell sample. cDNA was
generated using the High Capacity cDNA Reverse Transcription Kit (Life Technologies)
according to the manufacturer’s protocol. The generated cDNA samples were diluted with 100
17
μL of water and stored at -20°C. RT-qPCR assays were conducted using Taqman probes
(Applied Biosystems, Table 3). All gene expression levels were normalized to corresponding
expression levels of housekeeping gene 18S, which encodes for 18S ribosomal RNA
3. Investigation of DOX-induced intracellular iron accumulation in hiPSC-CMs
3A. Quantifying dose-dependent effects of supplementary iron on hiPSC-CM viability
Given that the hiPSC-CMs were normally cultured in medium depleted of ferric or
ferrous iron, it was first necessary to identify an optimal, nontoxic concentration of
supplementary iron that the cells could be pre-treated with. Non-DOX-treated hiPSC-CMs (D15-
20 post-differentiation) were harvested and re-plated onto Corning® 24-well plates. After 2-3
days or after beating was confirmed in each of the wells, hiPSC-CMs were treated for 24 hours
with 0, 1, 10, or 100μg/mL ferric citrate (Sigma-Aldrich) in regular CDM3 medium. The
CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was used to assess the dose-
dependent effects of iron supplementation on the viability and metabolic activity of hiPSC-CMs.
Briefly, this single-reagent, homogenous assay results in cell lysis and generation of a
luminescent signal (produced by the luciferase reaction) that is directly proportional to the
amount of ATP present within the cell—which, in turn, serves as an indicator of cell metabolic
activity and viability. Quantification of luminescence was conducted using a Cytation5 Imaging
Reader (BioTek) and the Gen5 2.07 software. As supporting qualitative data, Mitotracker®
Green FM (Life Technologies) was also employed to monitor the mitochondrial structural
integrity across the increasing iron concentrations.
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3B. Quantifying mitochondrial iron accumulation in DOX-treated hiPSC-CMs
Given the lack of assays that allow direct and specific detection of labile chelatable iron
(LCI) in living cells, researchers primarily rely on spectroscopic probes that allow real-time in
situ monitoring of LCI through one of two mechanisms: (1) reversible quenching of fluorescence
upon binding iron and other divalent ions or (2) generation of a fluorescent signal by the metal-
catalyzed oxidation of a fluorescent unit42. As an example of the first mechanism, rhodamine B-
[(1,10-phenanthrolin-5-yl)-aminocarbonyl] benzyl ester (RPA, Axxora, MW=834.8 g/mol), an
LCI-sensitive, mitochondria-specific red-fluorescent probe (ex: 564 nm, em: 601 nm), was used
to quantify mitochondrial chelatable (redox-active) iron levels in response to increasing
concentrations of DOX42,43. RPA is loaded into the mitochondria through potentiometric
distribution and, importantly, its fluorescence is stoichiometrically quenched by Fe2+ (ferric iron)
residing in the mitochondrial space42. Following a 24-hour co-treatment with DOX (1 μM, 3 μM
or control) and 5mg/mL ferric citrate, cells were treated with RPA (1 μM in HBSS) for 12
minutes, followed by three washes to fully remove excess RPA/RPAC and an additional 15-
minute incubation in HBSS. For each concentration of DOX tested (1 μM, 3 μM or control), an
iron-insensitive RPA control reagent (RPAC, Axxora) was first used to establish the baseline
fluorescence level. After normalizing to the DOX auto-fluorescence levels in each of the wells,
the difference in fluorescence between that of RPAC and RPA-treated cells (Δfluorescence) was
interpreted as the change in mitochondrial iron content, and, specifically, a reduction in
fluorescence as an indicator of mitochondrial iron accumulation. Quantification of fluorescence
and qualitative imaging was conducted using a Cytation5 Imaging Reader (BioTek) with Gen5
2.07 software and a Leica DFC500 fluorescent microscope with LAS X software, respectively.
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4. Statistical Methods
Data are presented as mean ± SEM. Comparisons were conducted via Student’s t-test with
significant differences (*) defined by p<0.05. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** IP ≤
0.0001 )
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RESULTS hiPSCs express key pluripotency markers and hiPSC-CMs express cardiac-specific sarcomeric
proteins
Human induced pluripotent stem cells (hiPSCs) formed colonies in vitro and expressed
standard pluripotency markers, including Oct3/4, Sox2, Nanog, and TRA-1-81 (Supplementary
Fig 1A, B). hiPSC-CMs were generated from all three hiPSC populations (HEALTHY
CONTROL, DOX CONTROL and DOX TOX) using a recently published, high-efficiency
protocol featuring a chemically defined differentiation medium (Fig 1B)36. Following
purification via glucose starvation, hiPSC-CMs formed beating layers and, as demonstrated by
immunofluorescence staining, expressed cardiac troponin (cTnT) and α -actinin, which form
intercalated patterns along sarcomeric Z-lines (Supplementary Fig 1C, D).
hiPSC-CMs demonstrate dose-dependent cytotoxicity in response to in vitro DOX treatment
HEALTHY CONTROL hiPSC-CMs were treated with increasing concentrations of DOX
(0, 0.1, 1 or 10 μM for 24 hrs, unless stated otherwise), and dose-dependent responses were
assessed using a variety of quantitative and qualitative methods (Fig 2A). After treatment with 0,
0.1, 5 or 25 μM DOX for 24 hrs, cardiac-specific immunofluorescence of hiPSC-CMs
demonstrated dose-dependent cytotoxicity, with visible loss of cell density and sarcomeric
integrity at 5 μM and 25 μM DOX (Fig 2B). The Mitotracker® Green fluorescent probe (Life
Technologies), which was used to visualize mitochondria in live DOX-treated hiPSC-CMs,
revealed a similar dose-dependent reduction in mitochondrial structural integrity (Fig 2C).
Beginning with the 1 μM treatment group, hiPSC-CMs exhibited visible and dose-dependent
condensation of the mitochondria, which usually assume a diffuse, punctate perinuclear pattern
21
(in healthy cardiomyocytes). Taken together, these visual data offer qualitative evidence of
DOX-induced cytotoxicity and apoptosis. This was further validated using the resazurin-based
PrestoBlue® Cell Viability Reagent and the JC-10 mitochondrial membrane potential assay, both
of which indicated dose-dependent reduction in cell viability and mitochondrial membrane
potential, respectively (Fig 2D, E). The reduction in cell viability and mitochondrial membrane
potential became statistically significant at the highest concentration (10 μM DOX), with little to
no alterations in either parameter at lower concentrations. Thus, 0.1 μM and 1 μM were
identified as relatively less cytotoxic concentrations at which we may study the early pathogenic
mechanisms that underlie DIC and precede DOX-induced cell death. Briefly, mitochondrial
membrane potential was quantified as a ratio of orange to green fluorescence, and thus,
decreasing relative ratios serve as an early indicator of apoptosis. Taken together, these results
suggest that hiPSC-CMs offer a viable and reliable in vitro system in which to study the
underlying mechanisms of DOX-induced cardiotoxicity.
Transcriptional analysis of ABCB8 expression in DOX-treated hiPSC-CMs
At approximately D15-20 after the start of differentiation, I determined gene expression
levels of ABCB8 in DOX-treated HEALTHY CONTROL, DOX CONTROL and DOX TOX
hiPSC-CMs. ABCB8 encodes for ATP-binding cassette subfamily B member 8 (ABCB8), a
transporter protein which localizes to the inner membrane of the mitochondria and is thought to
regulate the entry of iron into the mitochondrial matrix25. All ABCB8 expression levels were
normalized to corresponding expression levels of housekeeping gene 18S, which encodes for 18S
ribosomal RNA. The representative data for the above three patient-specific populations (Fig 3A,
B, C) indicate that DOX does, indeed, induce a dose-dependent downregulation of ABCB8
22
expression at high concentrations of DOX, as previously found in animal-based models26.
Transcriptional analysis revealed minimal changes in ABCB8 expression in the 0.1 μM DOX
treatment group and statistically significant reduction in ABCB8 expression in the 10 μM DOX
treatment group. While downregulation of ABCB8 was occasionally observed at 1 μM DOX (as
exemplified in Fig 3B), many of these alterations were of less or no statistical significance.
While all hiPSC-CM populations (HEALTHY CONTROL, DOX CONTROL and DOX TOX)
exhibited downregulation of ABCB8 at high concentrations of DOX, there were no apparent or
statistically significant patient-specific differences in the severity or onset of this transcriptional
change.
In addition, I investigated DOX’s effects on the expression of other iron metabolism
genes, focusing on three that were previously shown to be altered (either at the transcriptional or
protein level) in response to DOX in in vitro animal-based models26,44. The preliminary data for
HEALTHY CONTROL cells suggests dose-dependent downregulation of ACO1, which encodes
iron regulatory protein-1, or IRP-1 (Fig 3C). IRP-1 is a bifunctional protein that moderates
intracellular iron levels by binding to iron regulatory elements (IREs) of target mRNA and
regulating the expression of proteins involved in iron transport and storage44 (Supplementary Fig
2). Previous studies had demonstrated that DOX irreversibly inactivates IRP-1 by suppressing its
mRNA-binding ability, but the drug’s transcriptional effects on ACO1 were less well known44,45.
A similar dose-dependent down-regulatory trend was observed for SLC25A28, which encodes
mitoferrin-2, a transporter protein that facilitates the movement of iron into the mitochondria46
(Fig 3D, Supplementary Fig 2). Non-toxic concentrations of DOX did not produce significant
changes in the expression level of SLC40A1, which encodes ferroportin-1 (Fpn1, also known as
23
IREG1 or MTP1), a protein that facilitates the export of iron into the out of the cell47 (Fig 3E,
Supplementary Fig 2).
Transcriptional analysis reveals patient-specific variations in baseline expression levels of
ABCB8 and other key iron metabolism genes
In addition to DOX-induced alterations to ABCB8 expression, I investigated potential
patient-specific differences in the baseline gene expression levels of ABCB8. The data, both of
individual experiments (Fig 4A) as well as the average values across multiple experiments (Fig
4B), suggest that DOX CONTROL and DOX TOX hiPSC-CMs collectively express
significantly lower levels of ABCB8 relative to those generated from healthy individuals (DOX
CONTROL: n = 4, DOX TOX: n = 3). While some DOX TOX lines expressed ABCB8 at
significantly lower levels than select DOX CONTROL lines, this was not a robust pattern when
investigated across multiple experiments and patient-specific lines (Fig 4B). Interestingly,
relative to their HEALTHY CONTROL counterparts, DOX TOX cells also expressed reduced
levels of TOP2B, which encodes topoisomerase 2β (TOP2B), an analog of topoisomerase 2α
(TOP2A) that has previously been identified as a potential target of DOX and alternative
mediator of DIC21,22 (Fig 4C). Preliminary data also suggests a similar population-specific
difference in the mRNA levels of ACO1, albeit a less dramatic one than that observed for ABCB8
and TOP2B (Fig 4D). As stated previously, the gene product of ACO1, iron regulatory protein-1
(IRP-1), has also been hypothesized to be inhibited by DOX44. As with the difference in baseline
ABCB8 expression across the DOX CONTROL and DOX TOX populations, further
investigation is necessary to determine the robustness and implications of such variable gene
expression between HEALTHY CONTROL and two breast cancer patient-derived lines.
24
Application of iron fluorescent sensors to quantify mitochondrial iron accumulation in DOX-
treated hiPSC-CMs
Finally, rhodamine B-[(1,10-phenanthrolin-5-yl)-aminocarbonyl] benzyl ester (RPA,
Axxora), a mitochondria-specific fluorescent iron reporter, was used to quantify the level of iron
in the mitochondria of DOX-treated hiPSC-CMs. Since all hiPSC-CMs are normally cultured in
iron-depleted medium, it was first necessary to identify an optimal, nontoxic concentration of
supplementary iron that the cells could be pre-treated with prior to measuring intra-mitochondrial
iron accumulation in response to DOX. Visualization using Mitotracker® Green revealed dose-
dependent changes in mitochondrial structural integrity in response to ferric citrate, with visible
condensation of mitochondria (a visual sign of cytotoxicity) occurring in cells treated with 10
μg/mL ferric citrate and higher (Supplementary Fig 3A). When treated with a range of
concentrations of ferric citrate (Sigma-Aldrich) for 24 hours, hiPSC-CMs tolerated
concentrations of 1 μg/mL and 10μg/mL without significant reduction in metabolic activity
(Supplementary Fig 3B). Based on these two sets of data, 5μg/mL was selected as an appropriate
dose of ferric citrate that would likely serve as a sufficient iron pre-treatment while minimizing
any confounding iron-mediated cytotoxicity.
Following a 24-hour combined treatment of DOX (0, 1, 3 μM) and 5μg/mL ferric citrate,
hiPSC-CMs (D15-20 after the start of differentiation) from each condition were treated with
RPA or RPA control (RPAC), an iron-insensitive structural analog of RPA. For each DOX
concentration, the mitochondrial iron content was indirectly quantified as the difference in
fluorescence (∆fluorescence) between RPAC and RPA-treated cells (Supplementary Fig 3C).
Here, I investigated dose-dependent trends in %change of fluorescence, with the hypothesis that,
if DOX’s effects on iron metabolism increase mitochondrial iron accumulation, the % change in
25
fluorescence (relative to that of the untreated control) would be positively correlated with the
concentration of the in vitro DOX treatment. While a substantial reduction in fluorescence was
observed between RPAC and RPA-treated hiPSC-CMs across all concentrations, there was no
visually apparent dose-dependent trend in the degree of this “quenching” effect (Fig 5A). All
fluorescence values were normalized for DOX’s auto-fluorescence as well as to the RPAC
fluorescence of the untreated control group. Following these normalization steps, the data
demonstrated a clear reduction in RPA fluorescence at all concentrations of DOX and across all
three patient-specific hiPSC-CM populations (Fig 5C, E, G). Furthermore, the RPAC values
remained relatively consistent for all concentrations, thus ruling out variations in baseline
fluorescence as a confounding variable (Fig 5C, E, G). The preliminary data showed either no
significant dose-dependent change in the %change in fluorescence, or, unexpectedly, even a
decrease across increasing concentrations of DOX (Fig 5D, F, H).
26
DISCUSSION
Summary of main findings
This project applied hiPSC-CMs as a human in vitro model of DOX-induced
cardiotoxicity (DIC), specifically for the purpose of studying DOX-related alterations to iron
metabolism from diverse perspectives and experimental approaches. The experimental design
was constructed around two specific aims: to (1) validate findings from previous animal-based
models, namely ABCB8 downregulation and iron accumulation in response to DOX, in the
hiPSC-CM model and (2) whether the severity or pattern of either of these phenotypes can
inform and predict patient-specific susceptibility to DIC.
First, I validated the use of hiPSC-CMs as a viable in vitro model of DIC by confirming,
both qualitatively and quantitatively, that these cells are susceptible to dose-dependent
cytotoxicity in response to DOX. Next, I utilized qPCR to determine whether DOX’s effects on
ABCB8 expression, as observed in neonatal rat cardiomyocytes and in vivo murine models25,23,
could be recapitulated in this novel in vitro human context. Consistent with these previous
findings, the data suggested that DOX induces significant downregulation of ABCB8 at
concentrations such as 10 μM, a relatively high dose that is accompanied by significant reduction
in cell viability and mitochondrial membrane potential in hiPSC-CMs. However, “nontoxic”
concentrations of the drug, or more modest concentrations devoid of confounding effects on
overall cell health, were shown to have less significant effects on ABCB8 expression. These
findings suggest that, at least in this hiPSC-CM model, ABCB8 downregulation does not play a
key mechanistic role in the induction of oxidative stress and DIC, but is more likely to be a
byproduct of general cell death or a downstream effect of an alternative mechanism.
27
Despite my initial hypothesis that the DOX TOX hiPSC-CMs (derived from patients
positive for DIC) would demonstrate lower expression of ABCB8 than their DOX CONTROL
counterparts, this pattern was only observed between certain lines from the two populations and
was not significant in the average expression levels calculated across multiple experiments and
patient lines. Further studies are necessary to confirm the robustness of this pattern and
determine whether the level of baseline ABCB8 expression—and subsequently, the innate
efficiency of mitochondrial iron efflux and predisposition to excess ROS production—may serve
as a potential candidate for a biomarker to predict patients’ susceptibility to DIC. Interestingly,
the hiPSC-CMs derived from breast cancer patients (DOX CONTROL and DOX TOX)
exhibited significantly lower baseline expression of ABCB8 relative to that of the HEALTHY
CONTROL hiPSC-CMs. While this trend was considerably more robust than that between DOX
CONTROL and DOX TOX cells, additional studies are necessary to validate this pattern. This
novel finding raises the possibility that gene expression profiles with reduced ABCB8 expression
may predispose an individual to the development of breast cancer.
Finally, to explore the hypothesis that altered iron handling and mitochondrial iron
accumulation mediate DOX-related oxidative stress and cardiotoxicity, I employed fluorescent
iron sensors to detect and quantify the intra-mitochondrial iron content of DOX-treated hiPSC-
CMs. Despite my initial hypothesis that increasing DOX would lead to an increase in
mitochondrial iron content, the preliminary results indicated either no change or, in some cases, a
dose-dependent reduction in the degree of iron buildup. While further studies and repeat
experiments are required to validate both of these patterns, the latter finding may hint at a
confounding effect by DOX itself. Previous studies have demonstrated that iron promotes
oxidative stress partly through the formation of anthracycline-iron complexes7, and given DOX’s
28
preferential accumulation in the mitochondria26, it is possible that DOX’s iron-binding capacity
interferes with the interactions between iron and the RPA reagent. If DOX is competing with
RPA for iron in the mitochondrial compartment and preventing the iron from quenching the RPA
fluorescence, this may explain the apparent dose-dependent in the %change in fluorescence.
Implications of findings
Contrary to my hypothesis, DOX’s transcriptional regulation of ABCB8 expression was
revealed to be less significant and robust than suggested by in vitro and in vivo animal-based
studies25,26. This discrepancy in the findings may be attributed to differences in experimental
design, inherent species-specific differences, or both. First, unlike previous studies that subjected
their isolated animal cardiomyocytes to 10 μM and 20 μM DOX, my experiments included lower
concentrations of DOX that, importantly, had minimal effects on cell viability and thus may be
more likely to provide insight into the specific pathological mechanisms of DIC. Given that the
downregulation of ABCB8 was apparent at 10 μM but less so at the nontoxic concentrations, it is
possible that this is a byproduct of general cell death rather than a specific mechanism of ROS
production and DIC. Second, as previously discussed, animal-based in vitro assays are limited by
species-specific differences in cardiac physiology, which make it difficult to extrapolate its
findings to the pathology of drug toxicities and other disease conditions as it occurs in the human
body. The advantage of hiPSC-CMs specifically lies in their human origins and patient-
specificity, making them a potentially more accurate model of DOX-related cardiomyopathy and
the relative importance of various processes in causing cardiotoxicity. Thus, it is possible that,
while misregulation of ABCB8 may be a major effect of DOX in the rat and murine heart, these
transcriptional changes and subsequent disruption of iron metabolism play less significant roles
29
in DIC as it occurs in the human physiological context. In the latter case, these results serve to
highlight the inherent limitations of animal-based models and the advantages of humanized in
vitro studies in safely and reliably translating its findings into future clinical applications.
A particularly intriguing and unexpected discovery in this study was the difference in
baseline ABCB8 expression between the two breast cancer patient-derived hiPSC-CM lines
(DOX CONTROL and DOX TOX) and the HEALTHY CONTROL hiPSC-CMs. Although
ABCB8 and its gene product have recently been implicated in conferring doxorubicin resistance
in melanoma cells48, it was surprising to observe any difference in baseline ABCB8 expression
between cell populations that differed solely by whether the original patients had breast cancer or
not. This suggests that the role of ABCB8 may extend beyond mediating iron transport and
chemotherapy resistance, but perhaps also in tumorigenesis and proliferation of cancer cells.
Recently, the broader class of ABC transporters and its potential implications in the development
of cancer has become a topic of interest among researchers. Certain members of the ABC
transporter family are thought to play critical roles in enhancing cell proliferation and survival by
mediating cell-cell signaling via export of molecules such as leukotrienes, prostaglandins and
S1P, which go on to activate tumor-promoting lipid signaling pathways44. In the context of this
model, however, it would be expected that the cell populations derived from cancer patients have
relatively elevated expression levels of ABCB8 when compared to their HEALTHY CONTROL
counterparts, not lowered, as was observed in this work. Thus, while my findings support the
involvement of ABCB8 in cancer, they seem to contradict some of the ideas raised by recent
studies regarding this topic, and the specific mechanisms by which ABCB8 may affect the
clinical status of cancer patients is unclear. As evidenced by my preliminary findings and the
unanswered questions they evoke, elucidating the specific physiological roles of the ABCB8
30
protein—both in health and illness—is an essential step toward accurately identifying the role of
ABCB8 in DIC.
Future directions
While ABCB8 downregulation may not be significant at the mechanistically relevant
concentrations of DOX, alternative approaches should be taken to further elucidate potential ties
between DOX and the ABCB8 protein and their downstream effects. Previous findings in in
vitro and in vivo animal-based studies have suggested that disruption of ABCB8 expression
exacerbates cardiotoxicity through excess accumulation of iron and subsequent oxidative stress,
and, conversely, that these can be ameliorated through artificial overexpression of ABCB825,26. In
addition to using Western blotting to study ABCB8 protein levels in DOX-treated hiPSC-CMs, it
would be of great interest to employ lentiviral transfection and shRNA tools to overexpress and
repress ABCB8 expression, respectively. By observing the effects of such manipulation on
mitochondrial iron accumulation as well as other parameters such as cell viability, ROS
production and mitochondrial structural integrity, it is possible to glean more information about
the potential link between ABCB8 and mitochondrial iron overload, as well as the relative
importance of ABCB8 in mediating and exacerbating DIC.
Further optimization of the RPA-based assay and exploration of alternative methods of
intracellular iron detection are necessary to draw a more definitive conclusion regarding the
effects of DOX on mitochondrial iron content, as well as patient-specific differences—if any—in
the degree or onset of DOX-induced iron buildup. While the expected “quenching” of RPA
fluorescence was observed across all treatment groups and cell populations, practical challenges
and potential confounding variables limit the assay’s utility as a high-throughout platform with
31
which to study mitochondrial iron accumulation in hiPSC-CMs. As discussed previously, the
unexpected inverse relationship between the concentration of DOX and the mitochondrial iron
content (as determined by the %change in fluorescence) suggest potential interference by DOX
itself. If DOX preferentially localizes to the mitochondria and is somehow competing with the
RPA for the iron, this confounding factor makes it difficult to accurately quantify iron levels
using this assay. Second, since both RPA and RPAC are loaded into the mitochondria through
potentiometric distribution, the reliability of the data is contingent on the consistency of
mitochondrial membrane potential across all treatment conditions. Given DOX’s observed
effects on mitochondrial membrane potential (as demonstrated by the results of the JC-10 assay),
this limited the range of drug concentrations that could be tested. These practical obstacles
demand efforts to explore, develop and optimize alternative intra-mitochondrial iron detection
methods to circumvent such complications and better study the acute effects of higher
concentrations of DOX in an in vitro platform. Potential approaches include the visualization of
iron deposits using electron microscopy or treatment with Perl’s Prussian blue iron stain50, as
well as the use of radiolabeled iron followed by mitochondrial isolation and analysis using a
liquid scintillation counter26—all methods that have previously been applied in isolated animal
cardiomyocytes and other in vitro platforms, but have yet to be tested in hiPSC-CMs.
Despite the challenges accompanying the RPA-based assay, this work represents one of
the first attempts to study the phenomenon of iron overload and misregulation of iron transport,
as well as their consequences, in a hiPSC-CM model. Beyond its applications in the study of
DIC, the hiPSC-CM model may be applied toward the study of iron overload cardiomyopathy
(IOC) and other cardiac conditions caused by or involving impaired iron mobilization and
storage51,52. Patient-specific hiPSC-CMs may be employed as a sustainable platform for high-
32
throughput screening of existing chelating drugs and emerging therapies, as well as more
mechanistic investigations of the various conditions that fall under the umbrella of IOC. As
previously mentioned, such applications must be preceded by or occur concurrently with efforts
to troubleshoot existing methods and exploring novel techniques for in vitro iron quantification,
optimizing the protocols for use in the hiPSC-CM model.
Broader challenges and unanswered questions
In addition to these immediate, actionable next steps, the results of this thesis should be
considered in the context of broader challenges that complicate the research of DIC. The
mechanisms of DOX-induced oxidative stress and cardiotoxicity are likely not limited to the
disruption of iron metabolism, and are, instead, a complex interplay of various pathways and
cellular responses. In parallel to the work surrounding DIC in the context of iron metabolism,
Yeh, et al. and other groups have generated a body of evidence supporting an alternative
hypothesis, namely that DOX-mediated ROS production occurs primarily through the inhibition
of topoisomerase 2β (TOP2B), rather than through alterations to iron metabolism
(Supplementary Fig 4)21,22,8. An analog of topoisomerase 2α (TOP2A), DOX’s primary
chemotherapeutic target, TOP2B is highly expressed in quiescent, non-proliferative cells such as
cardiomyocytes and is thought to be involved in the maintenance of mitochondrial DNA
(mtDNA) transcription53. Thus, it is possible that DOX’s binding to and inhibition of TOP2B
serves as the primary mechanism of DIC, whereby the loss of TOP2B activity results in impaired
transcription of various key genes implicated in regulation of oxidative stress and mitochondrial
biogenesis8,22.
33
Studies involving dexrazoxane (DXZ), the only current FDA-approved cardioprotective
agent for anthracycline-induced cardiotoxicity, reveal findings that support both theories, making
it exceedingly difficult to determine which mechanism is predominantly responsible for DIC.
DXZ has been shown to selectively chelate mitochondrial iron in vitro26, but, as demonstrated by
Lyu, et al., the compound also seems to alter the configuration of TOP2B in a way that
effectively shields it from the binding of the anthracyclines54. Unfortunately, there is significant
controversy surrounding the clinical use of DXZ, as some studies have suggested that it limits
the on-target anticancer effects of DOX55. These challenges further underscore the need for
continued, rigorous investigation of the pathophysiology of DIC to identify innovative
approaches toward improving the drug’s clinical utility.
In the context of this thesis, the observed downregulation of multiple key iron
metabolism genes, including not only ABCB8 but also ACO1, SLC25A28 and SLC40A1, suggests
that DOX may have a broader and more far-reaching impact on cardiomyocyte gene
transcription than a specific role in ABCB8 expression. It is possible that, consistent with the
work of Yeh, et al., the primary target of DOX is the function of TOP2B, and ABCB8 is one of
the many genes whose transcription is misregulated due to the drug-induced inhibition of the
topoisomerase enzyme. In this model, the downregulation of ABCB8 and its impact on iron
metabolism serve as secondary effects of an upstream abnormality, rather than a specific target
of the drug itself.
Limitations of the hiPSC-CM model
As exemplified by this work, the advent of hiPSC technology has opened up novel
methods that allow us to circumvent the imperfections of animal models and study human
34
diseases in a humanized context. However, hiPSC-CM and other hiPSC-based platforms have
challenges of their own, including issues of (1) differentiation efficiency, (2) cell subtype purity
and (3) cell maturity. Although the efficiency of cardiac differentiation has improved
dramatically over the past few years, there is still considerable line-to-line, as well as patient-to-
patient, variability in the purity of the resulting cell populations. The presence of non-
cardiomyocytes, including smooth muscle cells and endothelial cells, pose a barrier to accurately
modeling tissue and cell type-specific mechanisms in cardiac diseases.
In addition to this persistent problem of differentiation efficiency, every differentiation
tends to yield heterogeneous populations of cells comprised of variable proportions of atrial,
nodal and ventricular-like cardiomyocytes, which are characterized by distinct molecular
profiles. For example, atrial and ventricular cardiomyocytes differentially express genes such as
MYL2, MYL7 and SLN, which encode ventricular and atrial isoforms of myosin light chain 2
(MLC2V, MLC2A) and sarcolipin, respectively56,57,58. While MLC2V has previously been used
to genetically select for ventricular subpopulations among human embryonic stem cell (hESC)-
derived CMs, there is currently no reliable cell surface marker that would allow us to isolate
specific hiPSC-CM subpopulations via fluorescence-activated cell sorting methods59.
Moreover, hiPSC-CMs exhibit ion channel structure and sarcomeric morphology
resembling that of fetal cardiomyocytes and, unlike mature adult CMs, fail to form T-tubules,
which are essential structures for action potential propagation, calcium influx and excitation-
contraction coupling29. Thus, while hiPSC-CMs may overcome specific-specific differences
between animal and human cardiomyocytes, the relative immaturity of these cells may pose an
equally difficult challenge in accurately modeling cardiac diseases and drug-induced
cardiotoxicity as they occur in the adult human heart.
35
Finally, the hiPSC-CM platform employed here is not immune to the broader challenges
that complicate all forms of in vitro disease modeling. Current in vitro models, hiPSC-CMs or
otherwise, are far from being able to recapitulate the complex physiology of the human body,
and thus, any conclusions gleaned from these models cannot readily be applied toward the
clinical context. As highlighted by these practical challenges, further efforts in the realms of iPS
cell reprogramming, cardiac differentiation and hiPSC-CM characterization are needed to
achieve an in vitro model that better recapitulates the human heart and the diseases that affect it.
Conclusions
As demonstrated by this work, hiPSCs and hiPSC-derived somatic cells provide a
powerful and innovative platform upon which to investigate not only the mechanisms underlying
human disease, but also those contributing to patient-specific susceptibility and resistance to
these conditions. As an example of this emerging approach, this work represents one of the first
studies to apply hiPSC-CMs in the context of DOX-induced cardiotoxicity, and to my
knowledge, the first to attempt modeling DOX-induced alterations in iron metabolism and
studying their potential links to the broader pathogenesis of DIC. While recapitulated at the high
concentrations of DOX tested in previous animal-based studies, I found that DOX does not
induce significant ABCB8 downregulation at nontoxic, mechanistically relevant concentrations.
Preliminary investigations of DOX’s effects on mitochondrial iron accumulation proved
inconclusive, with neither consistent dose-dependent trends nor robust patient-specific variations
in the patterns of iron accumulation. Further studies using alternative methods of quantifying
iron levels in a non-DOX-dependent, mitochondria-specific manner should help to improve the
sensitivity of our assays. The variable expression of ABCB8 between HEALTHY CONTROL
36
and breast cancer patient-derived lines suggest that patient-specific variations in gene expression
can be observed and quantified in an in vitro hiPSC-CM platform. Given the limited knowledge
regarding patients’ apparent susceptibility and resistance to DIC, this work presents an exciting
first step toward identifying the specific biological reasons behind such patient-specific profiles.
As a further application, this may allow us to identify specific biomarkers, phenotypic trends and
assays with which researchers and clinicians may predict the patient’s susceptibility to DOX-
induced cardioxicity, should he or she be prescribed the drug. While such endeavors will require
extensive further study, they may open up an exciting intersection between clinical oncology and
the emerging field of personalized medicine. In addition to designing less cardiotoxic analogs of
DOX and identifying cardioprotective reagents to be administered alongside the drug, this novel
application of hiPSC technology may empower us to optimize and tailor chemotherapy regimens
to the genetic constellation and biological predispositions of each individual patient.
37
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44
TABLES
Table 1. Cell lines and reprogramming methods
Cell Population Type Number of Patient-specific
lines
Cell Source Reprogramming Method
HEALTHY CONTROL 3 Peripheral blood
mononuclear cells
Sendaivirus DOX CONTROL 4
Dermal fibroblasts DOXTOX 4
Table 2. Primary antibodies used for immunofluorescence staining
Primary antibody Raised in Catalog Number
Source
Polyclonal Anti-Cardiac
Troponin (cTnT)
Rabbit ab45932 Abcam
Monoclonal Anti- α-Actinin
(Sarcomeric)
Mouse A7811 Sigma-Aldrich
Polyclonal Anti-Nanog Rabbit SC-33759 Santa Cruz
Biotechnology
Monoclonal Anti-Oct3/4 Mouse SC-5279 Santa Cruz
Biotechnology
Polyclonal Anti-Sox2 (Y-17) Goat SC-17320 Santa Cruz
Biotechnology
Polyclonal Anti-TRA-1-81 Mouse MAB4381 Millipore
45
Table 3. Summary of Taqman probes used for qPCR assays
Gene Symbol Species Gene Name ABI Assay ID
18S Human Eukaryotic 18S rRNA Hs99999901_s1
MYH6 Human Myosin Heavy Chain 6, cardiac muscle,
alpha
Hs00411908_m1
ACO1 Human Aconitase 1, soluble Hs00158095_m1
ABCB8 Human ATP-binding cassette, sub-family B
(MDR/TAP), member 8
Hs00894817_m1
SLC40A1 Human Ferroportin, solute carrier family 40
(iron-regulated transporter), member 1
Hs00205888_m1
SLC25A28 Human Mitoferrin, solute carrier family 25
(mitochondrial iron transporter), member
28
Hs00945861_m1
TOP2B Human Topoisomerase (DNA) II beta 180 kDa Hs00172259_m1
46
FIGURE LEGENDS
Fig 1. Schematic demonstrating the generation of hiPSC-CMs from patient-specific
hiPSCs.
(A) Skin or blood tissue samples were reprogrammed into human induced pluripotent stem cells
(hiPSCs) using Sendai virus-based methods. These cells were subsequently differentiated into
cardiomyocytes (hiPSC-CMs), which were then used to investigate the effects of DOX on
ABCB8 expression and intracellular iron accumulation, and to identify possible patient-specific
differences in these parameters that could predict susceptibility to DOX-induced cardiotoxicity
(DIC). (B) hiPSC colonies were differentiated into cardiomyocytes using a high-efficiency
protocol featuring a chemically defined medium. (C) hiPSC reprogramming and subsequent
differentiation allows the generation of patient-specific hiPSC-CMs from individuals and
patients with varying degrees of susceptibility to DIC. This offers a powerful tool to not only
circumvent the limitations of animal-based models and challenges associated with using primary
cardiac tissue, but also to identify the mechanisms underlying patient-specific susceptibility to
DIC.
Fig 2. hiPSC-CMs demonstrate dose-dependent cytotoxicity in response to in vitro DOX
treatment.
(A) hiPSC-CMs were treated with 0, 0.1, 1, or 10 μM DOX (for 24 h), unless indicated
otherwise. (B) Cardiac-specific immunofluorescence visually demonstrates dose-dependent
cytotoxicity in response to DOX. hiPSC-CMs were stained for DAPI (left column) and α-actinin,
a cardiac-specific sarcomeric protein (middle column). (C) The mitochondria of DOX-treated
hiPSC-CMs were visualized using Mitotracker® Green. Merged images are shown (Green =
47
Mitotracker® Green, Blue = DAPI). Dose-dependent cytotoxicity was also quantitatively
assessed by (D) measuring changes in cell viability and (E) monitoring changes in mitochondrial
membrane potential, where the ratio of orange/green fluorescence serves as an indicator of
relative mitochondrial membrane potential. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤
0.0001)
Fig 3. Transcriptional analysis of ABCB8 expression in DOX-treated hiPSC-CMs
Gene expression levels of ABCB8 were determined in (A) HEALTHY CONTROL, (B) DOX
CONTROL, and (C) DOX TOX hiPSC-CMs (D15-20 following start of differentiation) after
treatment with 0, 0.1, 1, or 10 μM DOX. Data shown here is representative for multiple
experiments as well as for at least two different patient-derived hiPSC-CM lines per population.
In HEALTHY CONTROL hiPSC-CMs, the relative ABCB8 expression levels (normalized to
that of the untreated control condition) were determined at 0.1 μM (n = 4), 1 μM (n = 5) and 10
μM (n = 6). In DOX CONTROL hiPSC-CMs, ABCB8 expression levels were determined at 0.1
μM (n = 4), 1 μM (n = 4) and 10 μM (n = 5). In DOX TOX hiPSC-CMs, ABCB8 expression
levels were determined at 0.1 μM (n = 3), 1 μM (n = 4) and 10 μM (n = 4). Here, ABCB8
expression levels were normalized to housekeeping gene 18S. Preliminary data is shown for the
expression of other iron metabolism genes (D) ACO1, (E) SLC25A28 and (F) SLC40A1, which
encode aconitase-1/iron regulatory protein-1, mitoferrin-2 and ferroportin-1, respectively. Here,
expression levels were normalized to 18S and corresponding MYH6 expression levels. (*P ≤
0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001)
48
Fig 4. Patient-specific hiPSC-CMs exhibit variable baseline expression levels of ABCB8,
ACO1 and TOP2B
Expression levels of ABCB8 (at the baseline, untreated condition) of HEALTHY CONTROL,
DOX CONTROL and DOX TOX hiPSC-CMs are displayed as (A) data from individual
experiments as well as (B) average relative values across multiple experiments. Preliminary data
is shown for the baseline expression levels of (C) ACO1 and (D) TOP2B, both of which also
encode proteins hypothesized to be targets of DOX-mediated cardiotoxicity. (*P ≤ 0.05, **P ≤
0.01, ***P ≤ 0.001, ****P ≤ 0.0001)
Fig 5. Application of iron fluorescent sensor to quantify mitochondrial iron accumulation
in DOX-treated hiPSC-CMs
Following 24 hr. treatment with 5μg/mL ferric citrate and DOX (0, 1, or 3 μM), hiPSC-CMs
were loaded with mitochondrial iron indicator rhodamine B-[(1,10-phenanthrolin-5-yl)-
aminocarbonyl] benzyl ester (RPA) or RPA control (RPAC), the iron-insensitive control
compound (1 μM in HBSS, 37° C). (A) At all concentrations of DOX, a clear reduction in
fluorescence was observed between RPAC- vs. RPA-treated cells, consistent with (B) the
%change in fluorescence quantified for varying concentrations of RPA. Preliminary data for the
relative fluorescence levels, normalized (1) for the auto-fluorescence of DOX and (2) to RPAC
fluorescence for the untreated control group, are shown for (C) HEALTHY CONTROL, (E)
DOX CONTROL, and (G) DOX TOX hiPSC-CMs. (D, F, H) The corresponding relative %
change in fluorescence is also shown to illustrate trends in the degree of mitochondrial iron
accumulation for the three patient-specific hiPSC-CM populations.
49
Supplementary Fig 1. hiPSCs express key pluripotency markers and hiPSC-CMs express
appropriate intracellular sarcomeric proteins.
Peripheral blood and skin samples were obtained from healthy individuals and DOX-treated
breast cancer patients, respectively, and these cells were reprogrammed using a Sendai virus
expressing OSKM (OCT4, SOX2, KLF4, and MYC). Confocal microscopy images of
representative hiPSC colonies demonstrate expression of pluripotency markers such as (A) Sox2
and Oct3/4, (B) Nanog and TRA-1-81. (C, D) Confocal microscopy demonstrates expression of
cardiac-specific cardiac troponin (cTnT) and α-actinin in hiPSC-CMs.
Supplementary Fig 2. Schematic of iron metabolism genes and their gene products.
Intracellular iron transport, mobilization and storage are a complex system comprised of
numerous key iron metabolism genes and the proteins that they encode. Of these and other
factors not included in this diagram, this study investigated DOX’s effects on the expression of
ABCB8, ACO1, SLC25A28 and SLC40A1, which encode ABCB8, aconitase-1/iron regulatory
protein-1, mitoferrin-2 and ferroportin-1, respectively.
Supplementary Fig 3. hiPSC-CMs demonstrate dose-dependent reduction in cell viability
and mitochondrial structural integrity in response to iron pre-treatment.
Cells were “challenged” with 0, 1, 10, or 100μg/mL ferric citrate (24 hr. treatment) in order to
identify a nontoxic concentration of supplementary iron that could be co-administered with DOX
to hiPSC-CMs before assessing their mitochondrial iron content. The effects of iron were
assessed using (A) Mitotracker® Green FM to visualize the mitochondrial structures of iron-
treated hiPSC-CMs and (B) the CellTiter-Glo® luminescence-based assay to assess the
50
metabolic activity (ATP production) in iron-treated hiPSC-CMs. (C) RPA, a red fluorescent iron
sensor, was used to indirectly determine the mitochondrial iron content in DOX-treated or
untreated hiPSC-CMs. Mitochondrial iron content was indirectly quantified by the difference in
fluorescence (Δfluorescence) between the cells treated with RPA and those treated with RPA
control (RPAC, an iron-insensitive structural analog of RPA).
Supplementary Fig 4. Two proposed models for the biological and molecular mechanisms
underlying DIC.
The phenotypes observed for TOP2B and ABCB8 knockout murine models, among other
experimental approaches, suggest that DOX induces cardiotoxicity by inhibiting the function of
the TOP2B enzyme and down-regulating the expression of ABCB8, respectively22,25. Both
models are supported by studies regarding the cardioprotective effects of dexrazoxane (DXZ),
which is hypothesized to (1) prevent DOX from binding to and inhibiting TOP2B and/or (2)
selectively chelate iron from the mitochondria of cardiomyocytes.
51
FIGURES
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SUPPLEMENTARY FIGURES
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