Project proposalScreening of 20 potential drugs stimulating differentiation of resident cardiac stem cellsEleftheria Mastoridou, Nicholas Kar Hong Fok and Tess Oude WeerninkAssignment 4 Summer School Cardiovascular Research August 2015

OverviewIntroductionHypothesisStudy design

In vitroIn vivoMaterials and budget

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IntroductionResident stem cell types

Types of resident stem cellsc-kit+ Sca-1+Side population cellsCardiospheres Islet1+

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Introduction Detection of resident stem cells

Detection of resident stem cellsexpression of transcription factors for early myocyte

lineagee.g Nkx 2.5GATA-4MEF-2 ·

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Introduction Detection of resident stem cells

●Several surface markers:○ c-kit○ Sca-1○ MDR1○ Isl-1

None of these markers are specific for CSCs; they are found in haematopoietic stem cells, but also in other cell types. Whether these cells are true cardiac stem cells remains to be ascertained.

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Introduction Detection of resident stem cells

●CSCs differ from other types of stem cells as they are typically

○Lin−

○CD34− ○CD45−

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...More specifically -c-kit+

c-kit antibodybiomarkers e.g GATA4/5, Nkx2.5, MEF2 .

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...More specificallyStem cell antigen-1 positive (Sca-1+)

Stem cell marker (Sca-1)Telomerase activityCardiac transcription factorsCD31-

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...More specificallySide population cells (SP)● Abcg2 (ATP binding

cassette transporter)● Stem cell markers (e.g

c-kit+,Sca-1+) ● CD31−

● CD34+

● CD45−9

Isl-1+ cells

● Transcription factor islet-1

● Factors of early cardiogenesis (e.g Nkx2.5, GATA 4)

Figure 2. Cardiac progenitor cell classes.

e specifically

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TestingTest if a drug stimulates proliferation of resident cardiac stem cells1. BrdU: Synthetic analog of the nucleoside thymidine

Incorporated into replicatory DNA in dividing cells Subsequently detected by anti-BrdU antibodies 2. · Measurement of the differences in myocyte size and comparing it to the size of the heart.

Immunofluorescence - IHC protocol procedure

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Testing3. Expression of Connexin 40 and 43

indicator of coupling and communication between differentiated CMPC

4. Expression of α-actin (SP cells)

5. Transcriptional regulators involved in cell-cycling e.g Notch TGF-β

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Introduction Stimulants of stem cell differentiation

In vivoTNF/TNFR2/Lin-28 signalling Growth factors (IGF-1/HGF)

In vitro5-azacytidineGrowth factors (TGF)

Vitamin COxytocinBMP6

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Introduction Assessment of stem cell differentiation

Undifferentiated state: Stem cell-specific genes (isl-1, c-kit) Cardiomyocyte predisposition transcription

factors (Nkx-2.5, MEF2C, GATA-4)

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Introduction Assessment of stem cell differentiation

Differentiated state:Genes for-

Sarcomeric structures (β-MHC, MLC2V, α-actinin)

Electrophysiological function (SCN5a, Ryr2)

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IntroductionAssessment of stem cell differentiation

Differentiated state:Spontaneous contraction

Ventricular cell-like action potentialsExcitation–contraction coupling

Sarcomeric structuresAdrenergic stimulation response

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Introduction Assessing performance of the heart

Assessing performance of the heartProlongation of action potential duration (APD)QTc lengtheningShort time variability

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Introduction Assessing performance of the heart

Clinical measures that indicate

heart improvement:

Reduced infarct size

LV dilation

Increased wall thickness

Increased ejection fraction

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Significantly improved end-

diastolic pressure

Developed pressureVentricular Contractility Assessment

(dP/dt)

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Hypothesis

The 20 potential drugs are able to stimulate new generation of cardiomyocytes by

resident stem cells

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In vitroIn vivoMaterials and budget

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Study Design Overview

Study Design In vitro - Source of cardiac tissue

Human heart auricle Human foetal heartCatheter-based small biopsy specimens

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Study Design In vitro - Isolation of stem cells

Clonogenic expansionMagnetic Cell Sorting (MACS) using Sca-1

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Study Design In vitro - Differentiation stimulation

Experiment model (repeated for drug 1-20, n=25)0 hrs Coat a 60-mm dish with 0.1% gelatin

Seed cells at a density of ±10,000 cells per cm2Add growth mediumIncubate cells at 37°C24 hrs Replace growth medium with drug 1 solution mediumDay 3 Refresh drug solution medium at a 3 day intervalWeek 2-4 Check for beating cells

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Study Design In vitro - Differentiation stimulation

Positive controlOptimize resident CSC differentiation5-azacytidine, TGF-β1, Vit C

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Study Design In vitro - Differentiation stimulation

Study Design In vitro - Differentiation stimulation

Negative controlRepeat experiment model without the addition

of drug solution medium

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Study Design In vitro - Confirmation of differentiation

Beating cells Expression of cardiac genes

Cardiac troponin T, β-myocin heavy chain and cardiac actinValidation of the procedure and time course

Sarcomeric structures stainingQuantification of differentiation

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Study Design In vitro - Confirmation of differentiation

Study Design In vivo

20 potential drugs20 groups of mice of n=151 control group n=15

Occlusion of LCA for 30 minAdminister drug/placebo

Drug to drug groupsPlacebo (PBS) to control group

Borst O, Ochmann C, Schönberger T, Jacoby C, Stellos K, Seizer P, Flögel U, Lang F, Gawaz M, Methods Employed for Induction and Analysis of Experimental Myocardial Infarction in Mice. Cell Physiol Biochem 2011;28:1-12 30

© 2001 Terese Winslow, Lydia Kibiuk

Study Design In vivo

MeasurementsHeart failure assessment

Prolongation of action potential duration (APD)QTc lengtheningShort time variability

HistologyInfarct size

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Study Design In vivo

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0-30 minutes Inducing MI

30 minutes Administering drug/placebo

14 days Heart failure assessment

30 days Heart failure assessment

45 days Sacrifice mice, perform histology

Time points

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NumberProfessor 3

PhD 5

MSc 10

Investigators

Investigators

Materials1. Mice2. Potential drugs3. Reagents

Stock solutions ( PBS, EDTA, buffers for cell isolation/staining)

Antibodies/antiagents and dilutionsGrowth factors (TGFβ1, bTGF) Enzymes (collagenase)

4. EquipmentCell culture set upCentrifuge tubesInverted light microscopeMicroscope slides and glasses

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Estimated Budget

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Mice experiments 315 (21 x n=15) 100.000

Drugs 20 10.000

Materials - 20.000

Number Costs (€)

Human heart auricle 4cm3 200.000

Drugs 20 10.000

Materials - 15.000

In vitro

In vivo

Total 320.000

(No) Questions?