project proposal stem cell therapy
TRANSCRIPT
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?