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Journal Club

SLIDE: The author


PhD student in stem cell biology and regenerative medicine.
11 papers
SLIDE: Viral Myocarditis
Coxsackie virus B3 is a RNA virus of the enterovirus genome. The RNA encodes a polyprotein and
VP1 is a structural mature protein that is easy to tag for immunofluorescence.
SLIDE:PathophysiologyofMyocarditis
The pathogenesis of viral myocarditis is often divided into three phases. During the acute phase of viral myocarditis,
viral infection (represented by the green pentagon) and replication in the heart cause myocyte necrosis and activation
of the hosts immune system esp, cytokine production by macrophages, including IL-1, IL-2, TNF and IFN. During this
phase, inflammatory signalling is largely protective to the heart.
Cytokine signaling then causes an influx of immune cells (first innate, then adaptive) marking the subacute phase
(few weeks to several months). These inflammatory cells engage in viral elimination but importantly contribute to
cardiac injury. Activated virus-specific T cells may target the host Ags. This causes impairment of contractile function.
SLIDE:AIM
Testing novel therapeutics on hiPSC-CMs also allows for the assessment of potential cardiotoxicities and druginduced arrhythmias, which are leading causes of drug withdrawal from the pharmaceutical market.
SLIDE:StemCells
Undifferentiated cells, capable of giving rise to mature cells.
Totipotent stem cells can give rise to every cell in the embryo.
Pluripotent stem cells can generate all cells in the human body.
Multipotent stem cells give rise to every cell type of one lineage.
Totipotent means it has the ability to become any type of cell. This can be the embryo, part of the yolk sac and part of
the placenta.
WHERE? We find it very early on in the zygote.
Pluripotent cells dont give rise to the yolk sac or the placenta. They just give rise to the cells of the embryo.
WHERE? Can be derived from the blastocyst.
In adults, we have multipotent cells which can give rise to a range of different cell types. These cell types are all
within one group:
E.g. Haematopoietic cells give rise to all the cells of the blood but they dont give rise to hepatocytes or neurons.
Instead, youll have neural stem cells which will give rise to all the cells of the brain.

SLIDE: iPSCs
Making pluripotent cells from adult cells.
iPSC derivation is typically a slow and inefficient process, taking 12 weeks for mouse cells and 34 weeks for
human cells, with efficiencies around 0.01%0.1%.
Induced pluripotent stem cells were first generated by Shinya Yamanaka's team at Kyoto University, Japan, in
2006.[1] Their hypothesis was that genes important to embryonic stem cell function might be able to induce an
embryonic state in adult cells. They began by choosing twenty-four genes that were previously identified as
important in embryonic stem cells, and used retroviruses to deliver these genes to fibroblasts from mice. The
mouse fibroblasts were engineered so that any cells that reactivated the ESC-specific gene, Fbx15, could be
isolated using antibiotic selection.
Transgenic expression of the Yamanaka Factors
Because differentiated skin cells do not naturally express Oct4, Sox2, Klf4 and c-Myc, scientists artificially express
these factors in differentiated cells. Scientists are able to artificially express genes in cells using transgenic strategies.
Transgenics involves the delivery of a sequence of genetic material (a transgene) into cells growing in a petri dish.
The transgene typically encodes for a protein that is not actively expressed in the host cell, but when the transgene is
delivered into the cell it becomes translated and the functional protein is artificially expressed.

There are a number of different ways to deliver transgenes into cells, including the use of retroviral vectors. Retroviral
vectors are modified viruses that deliver foreign genetic material into a cell. Once delivered into the cell, the genetic
material becomes integrated into the host cells genome for long-term expression of the transgene. In the first
reprogramming experiments, Yamanakas group used retroviral vectors to deliver Oct4, Sox2, Klf4 and c-Myc, into
skin cells growing in a petri dish. Skin cells that received all four transgenes soon began to artificially express Oct4,
Sox2, Klf4 and c-Myc protein. After 2-3 weeks of forced expression of the Yamanaka factors some skin cells
underwent genetic changes that caused them to turn into pluripotent embryonic-like stem cells.

SLIDE: hiPSC-CM
These hiPSC-derived cardiomyocytes (hiPSC-CMs) could be used to investigate the pathophysiology and molecular
mechanisms of acquired cardiac disorders such as viral myocarditis. Although other cell types such as HEK293T and
HeLa have been used to study coxsackievirus infection, these noncardiac cells are not ideal for modeling
cardiomyocyte infection in viral myocarditis. Other cardiac cells such as HL-1 mouse atrial tumor cells are
immortalized, proliferative cells that are not reflective of adult human cardiomyocytes.

SLIDE: Methods
1. Differentiation of hiPSC-CMs From hiPSCs
Lentiviral reprogramming was used to generate 3 hiPSC lines from skin fibroblasts of 3 healthy individuals in a 7-member family
cohort. An additional 3 hiPSC lines were generated with a previously published Sendai virus reprogramming protocol using
peripheral blood mononuclear cells from 3 healthy individuals.
These 6 hiPSC lines were differentiated into hiPSC-CMs using a 2-dimensional monolayer differentiation protocol and were
maintained in a 5% CO2/air environment as previously published. Briefly, hiPSC colonies were dissociated with 0.5 mmol/L EDTA
into single-cell suspension and resuspended in E8 media (Life Technologies) containing 10 mol/L Rho-associated protein kinase
inhibitor (Sigma). Approximately 100 000 cells were replated into 6-well dishes precoated with Matrigel (BD Biosciences). Next,
hiPSC monolayers were cultured to 85% cell confluency. Cells were then treated for 2 days with 6 mol/L CHIR99021 (Selleck
Chemicals) in RPMI plus B27 supplement without insulin to activate Wnt signaling and induce mesodermal differentiation. On day
2, cells were placed on RPMI plus B27 without insulin and CHIR99021. On days 3 to 4, cells were treated with 5 mol/L IWR-1
(Sigma) to inhibit Wnt pathway signaling and induce cardiogenesis. On days 5 to 6, cells were removed from IWR-1 treatment and
placed on RPMI plus B27 without insulin. From day 7 onwards, cells were placed on RPMI plus B27 with insulin until beating was
observed. At this point, cells were glucose-starved for 3 days with RPMI (no glucose) plus B27 with insulin to purify hiPSC-CMs,
because cardiomyocytes can selectively metabolize fatty acids as a source of cellular energy. After purification, cells were cultured
in RPMI plus B27 with insulin. When replating hiPSC-CMs for downstream use, cells were dissociated with 0.25% trypsin EDTA
(Life Technologies) into a single-cell suspension and seeded on Matrigel-coated plates. Matrigel resembles the complex

extracellular environment found in many tissues and is used by cell biologists as a substrate for
culturing cells. CM differentiation = 10 days
2. CVB3-Luc Infections and Antiviral Treatments
CVB3 expressing either Renilla luciferase (RLuc-CVB3) were obtained by placing the Renilla luciferase coding
sequences, followed by a 3CD cleavage site, between the 5 untranslated region and the P1 coding region. Luciferase
gene is the reporter of expression. Gene is attached to a regulatory sequence on the CVB3 genome.
Interferon 1 (IFN1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine stocks were dissolved in water. Before CVB3-Luc
infection, day 30 to 35 postdifferentiation hiPSC-CMs were pretreated with antiviral compounds for 12 hours unless noted
otherwise.

3. Gene Expression
For quantitative reverse transcriptase polymerase chain reaction, RNA was extracted with the miRNeasy kit (Qiagen).
Complementary DNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and real-time
polymerase chain reaction was conducted on an Applied Biosystems 7900HT Fast Real-Time PCR System. For additional gene
expression analysis, a DNA Microarray was used (Affymetrix).

1.

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface.


Scientists use DNA microarrays to measure the expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a genome.
Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-,
or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
4. Immunofluorescence
For indirect immunofluorescence HL-1 cells grown on gelatin/fibronectin-coated coverslips were fixed with 2.5% paraformaldehyde in PBS (pH
7.2) for 15 min at 4C and permeabilized with 0.1% Triton X-100 for 3 min at room temperature. The primary antibodies used for indirect
immunofluorescent staining were: monoclonal anti-desmin from Sigma (catalogue no. D1033), and mAbs MF 20 to sarcomeric myosin heavy
chain (MHC) (kindly provided by D. M. Bader, Vanderbilt University) and to ANF (kindly provided by C. C. Glembotski, University of
California, San Diego). The secondary antibody was either rhodamine- or fluorescein-conjugated goat anti-mouse IgG (Sigma). Cultures

were examined on a Leica confocal laser scanning microscope equipped with a Polaroid digital image capture system and recorded on Kodak
Gold 100 film. Images are printed in false color. Imaging was performed using a DMILLED microscope (Leica Microsystems) or a
Zeiss LSM 510Meta confocal microscope (Carl Zeiss AG) using Zen imaging software.

5. Bioluminescence Imaging
Quantification of CVB3-Luc proliferation on hiPSC-CMs pretreated with IFN1.

Day 30 to 35 postdifferentiation hiPSC-CMs were plated in RPMI plus B27 with insulin on Matrigel at a density of 40000 cells per
well of a 96-well plate. At the time of CVB3-Luc infection, 6 mol/L Enduren, an extended-duration coelenterazine (Promega), was
added. After infection, bioluminescence imaging was conducted using a Xenogen IVIS 100 Imaging System. Living Image software
(Perkin Elmer) was used for image analysis.

6. Cell Metabolism and Viability Assays


WST-1 assay (COLORIMETRIC ASSAY) quantifying cellular metabolic output and viability after treatment with increasing amounts of
IFN1.

WST-1 reagent (Abcam) was used to determine hiPSC-CM metabolism and viability after antiviral treatment. After 48-hour
treatment with antiviral compounds, 10 L of WST-1 reagent was added to 100 L RPMI plus B27 with insulin on day 30 to 35
postdifferentiation hiPSC-CMs. After 24 hours, a microplate reader (Promega) was used to quantify conversion of tetrazolium salt
WST-1 into formazan dye at 420 to 480 nm absorbance. Absorbance reading correlated directly with cell viability.

7. Ca2+ Imaging
Dissociated day 30 to 35 postdifferentiation hiPSC-CMs were reseeded in Matrigel-coated 8-well Laboratory Tek II chambers
(Nalge Nuc International) and were treated with 5 mol/L Fluo-4 AM and 0.02% Pluronic F-127 (Molecular Probes) in Tyrode
solution for 15 minutes at 37C. Cells were washed with Tyrode solution afterward. Ca2+ imaging was conducted using a Zeiss LSM
510Meta confocal microscope (Carl Zeiss AG) and analyzed using Zen imaging software. Spontaneous Ca2+ transients were
obtained at 37C using a single-cell line scan mode.

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca 2+
Fluo3,fluo4andtheirderivativesallexhibitlargefluorescenceintensityincreasesonbindingCa2+.Thefluorescenceintensity
increaseonCa2+bindingistypically>100fold.

SLIDE: Results cTnT


They showed that hiPSC were differentiated into cardiomyocytes. They showed this by confirming the
expression of cardiac-specific sarcomeric proteins i.e. the cTnT.
They then showed the expression of CAR as shown by the yellow arrows, which is necessary for
infection with CVB3.
SLIDE: Results Heterogenous Cell Pop
They also performed immunofluorescence on a heterogeneous cell population of hiPSC-CMs. MOI is the ratio of the
number of virus particles to the number of target cells present in a defined space. You would expect to see about 99%
of the targets infected with the virus.
Cardiac troponin Tpositive hiPSC-CMs were more susceptible to CVB3-Luc infection compared with
noncardiomyocyte, -smooth muscle actinpositive mesenchymal cells. A higher incidence of CAR in CMs may
explain the higher levels of VP1 fluorescence VP1 is a viral capsid protein, more VP1 means more viral infection.
Could have also done a panel of CAR tagged cells. Some studies show that in murine models and in humans with
little to no CAR expression, there is less or no virally caused myocarditis.
<CAR>
This protein was found to be expressed in various regions of the body including the brain, heart and more. However,
we found that CAR expression is significantly lower in hiPSC-CMs in comparison with adult human left ventricular
myocardium, perhaps reflecting the previously observed functional immaturity of hiPSC-CMs

SLIDE: Black
This indicates that hiPSC-CMs are extremely susceptible to coxsackievirus infection.

InaheterogeneousunpurifiedpopulationofhiPSCCMsafteralowefficiencycardiacdifferentiation,cardiac
troponinTpositivehiPSCCMsweremoresusceptibletoCVB3Lucinfectioncomparedwithnoncardiomyocyte,
smoothmuscleactinpositivemesenchymalcells
SLIDE: Brightfield
Brightfield images of hiPSC-CMs infected with CVB3-Luc (multiplicity of infection [MOI] 5) show the progression of cellular cytopathic effect
because of viral infection >24h.

A cytopathic effect was observed from 6h. (This is where you see a change in the host cell, once the
virus invades - it may be in the form of changes in cell morphology or cell physiology.)
Starting at 6 hours postinfection with CVB3-Luc MOI 5, cells displayed irregular beating patterns that became
increasingly erratic over time, culminating in the eventual cessation of beating after 12 hours of infection

The successful infection with CVB3 was confirmed by the increased expression of VP1, which is a
component of the viral capsid. By 8h, the CMs are overridden with VP1, where the cTnT, which is in
green is obscured.
SLIDE: Calcium
Calcium imaging of cells (n=12) infected with CVB3-Luc at MOI 5 for 7 hours showed a significant reduction in beating
rate and increases in calcium transient duration, time to transient peak, and standard deviation of transient intervals,
suggesting that CVB3-Luc infection results in disrupted intracellular calcium handling in hiPSC-CMs (Figure 2D).

SLIDE: Drugs
Ribavirin and PDTC were also effective in inducing a concentration-dependent reduction of CVB3-Luc proliferation on
hiPSC-CMs, because they are well-established inhibitors of viral replication.
Importantly, IFN1, ribavirin, and PDTC did not induce significant cardiotoxic effects on hiPSC-CMs.
We observed significant hiPSC-CM death after treatment with high concentrations of fluoxetine, which in our assay
reduced CVB3-Luc proliferation at 4 to 8 mol/L. Fluoxetine overdoseinduced arrhythmias have been reported
previously but not cardiac cell death, as we have shown here

SLIDE: IFN
Luminescence equates to the amount of proliferation of CVB3-Luc but you only see this in the presence of Enduren + Luciferase.
EnduRen Live Cell Substrate provides the ability to measure Renilla luciferase luminescence for at least 24 hours after substrate
addition, with up to tenfold higher signal-to-background ratios than wildtype coelenterazines.

As a negative control, the antibiotic PenStrep did not cause a significant reduction in CVB3-Luc proliferation in hiPSCCMs.
IFN1 was able to abrogate CVB3-Luc proliferation in a concentration-dependent manner and at very low
concentrations, as demonstrated previously. Using a DNA microarray, we found that IFN1 reduces viral proliferation
by priming hiPSC-CMs against viral infection and activating multiple genes associated with viral RNA and protein
clearance

SLIDE: Summary
Using this hiPSC-CM technology, we were able to study the mechanisms of CVB3 infection on human cardiomyocytes. We found
that hiPSC- CMs express the coxsackievirus receptor needed to internalize CVB3, and hiPSC-CMs are highly susceptible to CVB3
infection because the virus is able to proliferate rapidly and destroy the cells in a matter of hours. Notably, we used hiPSC-CM
technology in conjunction with a genetically modified strain of CVB3 that expresses luciferase, a bio- luminescent protein. This
allowed us to quantify viral proliferation on hiPSC-CMs and screen for the antiviral efficacy of a panel of antiviral compounds. The
CVB3/hiPSC-CM system that we established here could serve as a platform for discovering novel antiviral compounds that can
effectively treat patients with viral myocarditis.

Human embryonic stem cells


New development in 2014
hSCs were grown in culture and converted to cardiomyocytes using GFs
Need billion cardiomyocytes
Cells had fluorescent Ca indicator introduced for recognition and to check functional integration
o Genetically encoded Ca indicator
o Can see Ca transients
Converted billion myocytes injected into margins of infarct
Myocytes integrated with existing myocardium and contracted
o Can tell because the fluorescent cells were integrating
Myocytes integrated with existing myocardium and contracted
Major development because in primates and myocytes showed functional integration
Problems:
Caused minor arrhythmias
Immune problems inevitable
Long run solved by iPSCs
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