welcome to the slas 2015 tutorial - cisbio bioassays · welcome to the slas 2015 tutorial presented...
TRANSCRIPT
1
Welcome to the
SLAS 2015 Tutorial presented by
February 10th, 2015
Coby Carlson Francois DegorceCarl Peters
CLARIOstar Highlights:
• Linear Variable Filter Monochromator The only adjustable bandpass monochromator
• Linear Varible Dichroic The only reader with an adjustable dichroic
• Monochromator, Spectrometer, Filters Three key technologies in one reader
• Superb luminescence dynamic range
• Two reagent injectors
CLARIOstar
Wavelength
Tra
nsm
itta
nce
Adjustable bandwidth
Adjustable peak wavelength
Wavelength
LVSWP LVLWP
Tra
nsm
itta
nce
*Angle of the arrow representing the light beam relative to the LVFs
is not Indicative of propagation angle or polarization of the light beam
LVF1LVF2
BMG‘s Linear Variable Monochromator is Different: Continuosuly Adjustable Bandpasses up to 100nm
Dichroics prevent excitation and unwanted light from reaching the
detection stage of the instrument. They are very important for optimal
sensitivity, but traditional dichroics have a fixed cutoff wavelength.
Without an adjustable dichroic a reader has to rely on the user having
the proper dichroic installed, or in worst case a 50/50 beam splitter
which reduces sensitivity.
BMG‘s Linear Variable DichroicThe Only Reader with an Adjustable Dichroic
ExcitationSource
DetectionSystem
Well
505LP Dichroic485nm
520nm
CLARIOstar
Technical features for performing HTRF
Optics:
Enclosed free air optical light path• 0.1 mm focus precision
• All assays top or bottom reading
CLARIOstar
CLARIOstar Technical Features
Top and Bottom Reading, Focus & Optical FlexibilityIf an assay can be read from the top, it can be read from the bottom on the CLARIOstar
and can focus with 0.1 mm precision which is key for detecting monolayers of cells. This
includes polarization, time-resolved fluorescence, luminescence.
High-resolution well scanning of adherent cells Z Axis
Adjustment
Liquid
level
CLARIOstar
Technical features for performing HTRF
Filters:
Ex / Em & dichroic slides• Easy to Change
• Identified using filter scan
Exictation Source:
High performance Xenon Flash Lamp
Detection System:
Photo Multiplier Tube (PMT)
CLARIOstar
Technical features for performing HTRF
TR-FRET
HTRF• TR-FRET measurement based on Cisbio chemistry
• Cisbio set some instrument quality limits which the CLARIOstar achieves
Delta F
Low 3 h
Delta F
high 3 h
Delta F
low 21 h
Delta F
high 21 h
PHERAstar
mean of 622 965 25 1100
CLARIOstar
(gain 2600)20 751 30 880
Omega
(gain 2600)Not meas. Not meas. 29 713
Cisbio Limit > 10 > 500 > 15 > 600
Please note that the CLARIOstar is HTRF certified for black and white plates.
Special Thanks to:
Team of German Engineers
BMG LABTECH Application Scientists
CDI and Cisbio
COMBINING HTRF DETECTION
TOOLS TO IPS CELL MODELS
MOVING TOWARDS MORE
BIOLOGICAL RELEVANCY
SLAS Washinghton D.C., US
Februray, 2015
1320 February
2015
Supporting all drug discovery steps with advanced assays
Cell signaling research
Drug discovery at all stages and all therapeutic areas
Standardized HTSComplex biological approaches
Unlimited applications
Biotherapeutics
Small molecules
AD & HTS
Hit-to-lead
TA
HTRF/iPS cells tutorial SLAS 2015
1420 February
2015
How Cisbio pioneered and refined cell-based TR-FRET
approaches
HTRF/iPS cells tutorial SLAS 2015
Cell and
biology
Add&read
Process
1520 February
2015
Efficient assay readout require optimal detection devices
Highly performing excitation sources (high energy flashlamp, laser)
Very selective signal collection and filtration (PMTs)
Time-resolved fluorescence mode
HTRF/iPS cells tutorial SLAS 2015
1620 February
2015
Custom assay
development services
An ever growing portfolio of cell-based tools
Ready-to use kits
Phospho-
protein
assays
Cytokine
assays
HTRF/iPS cells tutorial SLAS 2015
1720 February
2015
Creating efficient detection tools – enabling more
biological relevancy
HTRF/iPS cells tutorial SLAS 2015
Artificial Physiological
BiochemicalCell-based
(over-expressed)
Cell-based
(endogenous
primary, 3D, iPS)
18
Presentation Outline
• Introduction to platform technologies / providers
• Brief overview of iPSC technology and CDI
• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas:
• Diabetes and Alzheimer’s Disease
• Summary and Final Thoughts
19
iPS Cell TechnologyRevolutionary Access to Human Biology
Differentiate into
all 208 cell types in
the human body
208 C
ell
Typ
es
Represent
any individual
genotype
6 Billion People
Reprogramming
Dif
fere
nti
ati
on
Mastery of all three
technology platforms
is evolutionary,
and promises to
reinvigorate drug
discovery
20
iPS Cell TechnologyOvercoming Current Limitations
Primary Human Cells Transformed Cell Lines Animal Models
• Limited availability
• Variable
• Unstable
• Limited characterization
• Poor recapitulation
• Limited key functional
characteristics
• Cannot represent human
diversity
• Represents animal biology,
not human biology
• Resource intensive
• Large cmpd requirements
• Animal welfare issues
Induced pluripotent stem cell (iPSC) technology overcomes limitations of
existing cell models – Availability, Functionality, Reproducibility, Translatability
21
CDI Overview and Product Portfolio
2009 2010 2011 2012 2013 2014
iCell Cardiomyocytes iCell
Endothelial Cells
MyCell Products
iCell
Hepatocytes
iCell Astrocytes
iCell
Hematopoietic
Progenitor
Cells
iCell Skeletal
Myoblasts
iCell Cardiac
Progenitor CellsiCell
DopaNeurons
iCell Neurons
CDI is the world’s largest producer of human
iPSC-derived cell types
Headquartered in Madison, WI
Currently employs ~138 total staff
~650 yrs human stem cell experience
Core competencies
Creation and culture of human iPS cells
Genetic engineering of iPS cells
Development of new differentiation protocols
Manufacture of human iPS cell-derived cell types
iCell Products = normal healthy donor
MyCell Products = represent patient-
derived or a disease-specific donor iPS
cell line
iCell
Macrophages
22
iCell Products and HTP Screening?
Quality
Quantity Purity
Drawnel, et al. “Disease modeling and phenotypic
drug screening for diabetic cardiomyopathy
using human induced pluripotent stem cells”
Cell Reports. 2014; 9: 1–11.
Xu, et al. “Prevention of β-amyloid induced toxicity
in human iPS cell-derived neurons by inhibition of
cyclin-dependent kinases and associated cell cycle
events” Stem Cell Research; 2013;10: 213–227.
23
Presentation Outline
• Introduction to platform technologies / providers
• Brief overview of iPSC technology and CDI
• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas
• Diabetes and Alzheimer’s Disease
• Summary and Final Thoughts
24
ELISA vs. HTRF Comparison“Real-world” CDI example
iCell Astrocytes
Liang et al. (2004) J. Neurochem.
What can they do?
LXR agonist (T0901317)
stimulates ApoE expressionLiterature reference?
WB-to-ELISA conversionELISA-to-HTRF conversion
Standard Curve ELISA data
The minimum detectable dose (ie. the “sensitivity”)
of Apolipoprotein E is typically ~0.03 ug/mL
HTRF Assay Data for ApoE releasein human iPSC-derived Astrocytes
-10 -9 -8 -7 -6 -50
5
10
15
20
25
30
Log [T0901317] (M)
[Ap
oE
] (n
g/m
L)
Develop an application for ApoE secretion
Standard Curve
25
HTS-Compatible Format = Lots of Data“Bang for your buck”
iCell Macrophages
• M1 or M2 activation?
• Cytokine release assays
• Matrix experiments to define
IL-6 HTRF assay data
Several kits have been tested:
• TNF-alpha
• IL-1b
• IL-6
• IL-10
• IL-12
26
Cardioprotection
iCell Cardiomyocytes
Activation of AKT signalingin iCell Cardiomyocytes
(HTRF pAKT assays)
-13 -12 -11 -10 -9 -8 -7 -60
500
1000
1500
2000
2500
3000
-100
0
100
200
300
400
Thr308
Ser473
Log [IGF-1] M
Delt
a F
%
Delta
F%
Normoxia Hypoxia0
10
20
30
40
50
*
% A
po
pto
tic C
ells
(TU
NE
L/D
AP
I)
AB C
D
EDAPI
TUNEL
Normoxia Hypoxia
Control IGF-1 Insulin0
25
50
75
100
125
*****
Casp
ase-3
/7 A
ctiv
ity
F
27
Presentation Outline
• Introduction to platform technologies / providers
• Brief overview of iPSC technology and CDI
• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas
• Diabetes and Alzheimer’s Disease
• Summary and Final Thoughts
28
Diabetes and Metabolic DiseasesControlling Blood Glucose
GLUCOSE
MUSCLE
LIVER
ADIPOCYTES
β-cells α-cells
PANCREAS
INTESTINE
High blood sugar
= insulin released
= glucose taken into cells
Low blood sugar
= glucagon released
= stimulate gluconeogenesis
29
Diabetes and Metabolic DiseasesUse of iPSC-derived cell types to study disease
iCell Hepatocytes
iCell Skeletal Myoblasts
iCell Cardiomyocytes
iCell Neurons
iCell Endothelial Cells
GLUCOSE
MUSCLE
LIVER
ADIPOCYTES
β-cells α-cells
PANCREAS
INTESTINE
30
Insulin-stimulated Pathway Analysis
iCell Hepatocytes
Inhibition of AKT signalingin iCell Hepatocytes
(HTRF pAKT Ser473 assay)
-10 -9 -8 -7 -6 -5 -40.0
0.4
0.8
1.2
1.6
IC50 = 254 nM
Log [PI-103] (M)
HT
RF
Rati
o
(665 n
m /
620 n
m)
Activation of AKT signalingin iCell Hepatocytes
(HTRF pAKT Ser473 assay)
-11 -10 -9 -8 -7 -6 -50.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
Log [Insulin] (M)
HT
RF
Rati
o
(665 n
m /
620 n
m)
High blood sugar = insulin released = glucose taken into cells
Low blood sugar = glucagon released = stimulates gluconeogenesis
31
Glucagon-mediated Cell Signaling Events
iCell Hepatocytes
High blood sugar = insulin released = glucose taken into cells
Low blood sugar = glucagon released = stimulates gluconeogenesis
32
Modulating PI3K/AKT Signaling in iCell SkM
iCell Skeletal Myoblasts
33
Hot Target in Metabolism – PCSK9
iCell Hepatocytes
0.001
0.010
0.100
1.000
GAPDH HNF4A LDLR PCSK9
Re
lative
Ge
ne
E
xp
ressio
n
• PCSK9 regulates cholesterol levels in the blood
• One of the key drivers of high LDL cholesterol
• PCSK9 binds to LDL receptor, targets it for destruction
• If PCSK9 does not bind, LDLR can remove cholesterol
• Drugs that block PCSK9 can lower cholesterol
http://www.broadinstitute.org/
34
iPSC-based Alzheimer’s Disease Model
Genotype Phenotype Catalog #
Control (WT) Healthy / normal 01279.107
APP A673T (homo) Isogenic
AD model
01279.A27
APP A673V (het) 01279.A32
iCell Neurons MyCell
Disease and
Diversity
Products (DDP)
iPS Cells
Genetic Engineering
Human Neurons
β-secretase -secretaseAβAPP
Ala→Thr = protective and Ala→Val = causativeA673
A673T• 1st variant associated with protection
• Identified in a whole genome seq.
project of ~1,800 people from Iceland
• Jonsson et al. (2012) Nature
A673V• Also near BACE1 cleavage site
• Mutation contributes to AD pathology
• Increased Aβ production, and
enhanced aggregation and toxicity
• Di Fede et al. (2009) Science
MyCell Products
35
Genotype Impacts PhenotypeBiomarker Levels Differ From WT
DIV 20 4
Thaw and
Plate Cells
1
Change
Media
3
Run HTRF
Assay
Collect
Media
Factors that impact biomarker detection:
• Cell density
• Media composition
• Time in culture
• Dilution factor
Maloney, et al. “Molecular mechanisms of
Alzheimer’s disease protection by the A673T allele
of APP” J. Biol. Chem. 2014; 289: 30990-31000.
36
Presentation Outline
• Introduction to platform technologies / providers
• Brief overview of iPSC technology and CDI
• HTRF assays with iPSC-derived cell types• Focus on therapeutic areas
• Diabetes and Alzheimer’s Disease
• Summary and Final Thoughts
37
HTRF technology can address relevant biology
HTRF is a well-established assay technology in the drug discovery community
CLARIOstar has been validated for use with HTRF assays
iPSC technology is changing the way we study human disease
CDI has Applications and Support team to help ensure success
Development of HTS-compatible assays demonstrates possibilities for screening
with relevant cell types (secondary screens in TA most likely)
Quality of the data matters – good cells, good assays, good instrument for
detection are all a must!!
Develop applications with normal cells – and we are then poised to compare
assay performance with disease-specific or patient-derived cells
Phrases like “Human brain in a dish” and “in vitro clinical trials” are real
38
Thank you !!
CDI
Please visit us at our Booth:
BMG Labtech #929
Cisbio #923
CDI #1304