Valérie DEFFONTAINE, R&D Scientist, Quality Assistance Webinar held on 8th June 2017. The discovery of human pluripotent stem cells 10 years ago turned the spotlight on the potential of pluripotent stem cells for personalised cell therapy. The scientific interest then quickly shifted towards the use of these cells for safety pharmacology, drug discovery and disease modelling. For all these purposes, in the mid to long term, properly characterised cell banks will be necessary.  The characterisation of embryonic (ESC) and induced pluripotent stem cells (IPSC) used for manufacturing requires the development and validation of analytical methods (e.g. flow cytometry, microscopy, QPCR and bioassays). Cell characterisation includes the testing of cell product identity, determination of impurities, and assessment of biological activity and viability. Among the techniques available, flow cytometry is widely used to assess the expression of cell markers. Our laboratory has developed flow cytometry panels dedicated to the characterisation of extracellular and intracellular markers of ESC and IPSC, and to the detection of cell-related impurities. We proposed a method for the validation of flow cytometry panels according to the recommendations of international guidelines on the validation of analytical methods. IPSC differentiated into cardiomyocytes and MSC-like cells were also used to test the performance of our flow cytometry panels to accurately monitor the manufacturing process of cell products. In addition to the technical tips, this webinar aims at presenting a critical view on the use of flow cytometry platform for cell characterisation.  For more information, visit http://www.quality-assistance.com/analytical-services/CBMPs

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Valérie Deffontaine
08/06/2017
Characterisation of human
pluripotent stem cells (ESC & IPSC)
and by-products

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Summary
1. Introduction: ESC and IPSC in the cell therapy area
2. Focus on the analytics and characterisation methods
3. Characterisation of ESC/IPSC and by-products (MSC
and cardiomyocytes like cells)
4. We know…we share: observations from our analytical
experiments

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ESC and IPSC in the cell therapy area
EMBRYONICSTEMCELLSINDUCEDPLURIPOTENT
STEMCELLS
Skin Fibroblasts
Blastocyst

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ESC and IPSC in the cell therapy area
DIFFERENTIATION
CELL-BASED THERAPY
AUTOLOGOUS ALLOGENIC

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ESC and IPSC in the cell therapy area
Modified from Bionest Partners, 2010
Personalised cell
therapyDisease modelling
Drug discovery
Safety pharmacology
11 years after the discovery of IPSC…the goal has shifted!

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ESC and IPSC in the cell therapy area
Adapted from Bionest Partners, 2010

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ESC and IPSC in the cell therapy area
« IPS cells are only 10 years old. The research takes time. That’s
what everybody needs to understand » Shinya Yamanaka, Nobel
Prize Physiology Medicine 2012.
« The greatest future challenges are not scientific. Researchers are
going to need strong support from the pharmaceutical industry »
Edward Stevens, Pfizer Neuroscience and Pain Research Unit, UK.
Manufacturing Analytical
Services

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Analytics and characterisation methods
Some definitions …
Analytics: science that seeks to the improvements of
substances (e.g. chemical) or entities (e.g. cell)
composition measurements and corresponding data
interpretation.
Validation: demonstrates that a method will provide
accurate, precise and reproductible data during the
study-sample analysis.

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Analytics and characterisation methods
Selection of
validation
parameters
(validation or
qualification?)
Validation/
qualification
Pre-
validation/
qualification
System
Suitability
Tests
Development phase

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Analytics and characterisation methods
CELL CHARACTERISATION
Identity
Purity/Impurities
Biological activity
(potency)
Viability
Mass
spectrometry
Microscopy
ELISA
Cell-based
assays
PCR
FACS
Microarrays
ECL
(MSD)
Luminex

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Analytics and characterisation methods
CELL CHARACTERISATION
Identity
Purity/Impurities
Microscopy
PCR
FACS

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ESC/IPSC characterisation
IPSC
ESI-017 Human Embryonic Stem Cells line (ESI BIO)
Human IPS Cells line (Applied StemCell)
ESC

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ESC/IPSC characterisation
Method
Parameters
per sample
21 CFR part
11 compliant
software
Quantity of
cells per test
Time per
run
(analytics
+ process)
Flow
Cytometry
Quantitative +++ Yes Medium 2 days
QPCR
Semi
quantitative
++ Yes Small 1 day
Leica
Microscope
Mostly
qualitative
+ No High 3 to 4 days
BD FACSVerse™ &
FACSSuite™ software
Applied Biosystem™ 7500 Fast Real-
Time PCR & AccuSEQ™ software
Leica DMI6000B & Leica
Application Suites software

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ESC/IPSC characterisation
Validation versus
Qualification
Reference material
Qualified cell bank
(marker expression
= 100%)

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ESC/IPSC characterisation
Flow cytometry
TRA-1-60
SSEA-4
ALP
Rationale for the pluripotency panels design (Human)
SSEA-1
CD13
Sox2
Nanog
Oct3/4
Extracellular Intracellular
ESC/IPSC + ESC/IPSC - Original tissue

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ESC/IPSC characterisation
Flow cytometry
Validation workflow (Positive markers)
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ES/IPS cell banks
Labelling + Washing
ES/IPS ES/IPS
0.5*106c/ml
Accuracy, linearity (spikes, independant preparations)
1*106c/ml
0.5*106c/ml
Labelling plate
With antibodies cocktail
Without antibodies
cocktail
Analytical plate

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ESC/IPSC characterisation
Flow cytometry
Validation workflow (Negative markers)
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Positive cell line
banks
Labelling + Washing
Positive
cell line
0.5*106c/ml
Accuracy, linearity (spikes, independant preparations)
1*106c/ml
0.5*106c/ml
Labelling plate
With antibodies cocktail
Without antibodies
cocktail
Analytical plate
Positive
cell line

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ESC/IPSC characterisation
Flow cytometry
Validation of intracellular panel on ESC
Spike 50:
50% labelled and 50%
unlabelled cells
45.83 % 47.24 % 47.30 %
Nanog Oct3/4 Sox2
Nanog Oct3/4 Sox2

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ESC/IPSC characterisation
Flow cytometry
Validation of intracellular panel on ESC: linearity

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ESC/IPSC characterisation
Flow cytometry
Validation of intra panel on ESC: accuracy
Nanog Oct3/4 Sox2

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ESC/IPSC characterisation
Flow cytometry
Results: ESC & extracellular panel
98.96 %79.22 % 99.88 %0.05 %0.01 %
SSEA-1 CD13 TRA-1-60 SSEA-4 ALP
SSEA-1 CD13 TRA-1-60 SSEA-4 ALP

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ESC/IPSC characterisation
Flow cytometry
Results: ESC & intracellular panel
99.12 % 99.49 % 99.95 %
Nanog Oct3/4 Sox2
Nanog Oct3/4 Sox2

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ESC/IPSC characterisation
Flow cytometry
Results: IPSC & extracellular panel
98.12 % 91.20 %94.25 %0.05 %10.20 %
SSEA-1 CD13 TRA-1-60 SSEA-4 ALP
SSEA-1 CD13 TRA-1-60 SSEA-4 ALP

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ESC/IPSC characterisation
Flow cytometry
Results: IPSC & intracellular panel
95.75 % 97.15 % 99.72 %
Nanog Oct3/4 Sox2
Nanog Oct3/4 Sox2

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IPSC by-products characterisation
Study 1: Differentiation of IPSC in MSC-like cells
Step 1: Cells plating on 0.1% gelatin coated dishes in mTeSR 1 medium.
Step 2: Derivation step in 50% mTeSR 1 and 50 % derivation medium (alpha MEM,10% FBS,50 µM
magnesium L-ascrobic acid,100 nM dexamethasone).
Step 3: Derivation step in 100% derivation medium.
Step 4: Expansion step in expansion medium (20% O2) (Alpha MEM,10% heat inactivated FBS,1X
non essential amino acid,200 mM L-glutamine) or CellGro medium + FGF2 (3% O2).

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IPSC by-products characterisation
Flow cytometry
CD44
CD90
CD105
CD45
MSC (Extracellular) IPSC (Extracellular)
Pos Markers Neg Markers
Differentiation of IPSC in MSC-like cells
CD34
ALP
EpCam
TRA-1-60
SSEA-1

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ESC/IPSC: by-products characterisation
Flow cytometry
Differentiation of IPSC in MSC-like cells
Trend

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ESC/IPSC: by-products characterisation
Flow cytometry
Differentiation of IPSC in MSC-like cells
Trend

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ESC/IPSC: by-products characterisation
Flow cytometry
Differentiation of IPSC in MSC-like cells
Trend

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ESC/IPSC: by-products characterisation
Flow cytometry
Differentiation of IPSC in MSC-like cells
Trend

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IPSC by-products characterisation
Ventricular cardiomyocytes
(Cor.4U®)
Study 2: IPSC-derived cardiomyocytes (Axiogenesis)
Ventricular, atrial and
pacemaker cardiomyocytes
(CorV.4U®)
Cardiac fibroblasts
(FibroCor.4U®)

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IPSC by-products characterisation
Flow cytometry
Pluripotency
(Extracellular)
Pluripotency
(Intracellular)
Identity
Impurities detection
IPSC-derived cardiomyocytes
TRA-1-60
SSEA-4
ALP
SSEA-1
CD13
Sox2
Nanog
Oct3/4
Cardio
(Intracellular)
Troponin
Actinin
Myosin

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iPSC by-products characterisation
Flow cytometry
Ventricular
cardiomyocytes
Ventricular, atrial
and pacemakers
cardiomyocytes
Cardiac fibroblasts
iPSC-derived cardiomyocytes: Identity of cardiac cells
Cor.4U
CorV.4U
FibroCor.4U
Actinin TroponinMyosin

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IPSC by-products characterisation
Flow cytometry
Individual
screening of 8
markers
iPSC-derived cardiomyocytes: Cell impurities in cardiomyocytes

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IPSC by-products characterisation
Flow cytometry
Individual
screening of 8
markers
iPSC-derived cardiomyocytes: Cell impurities in cardiomyocytes
Multipotent cells? Pluripotent cells? Precursor cells?

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IPSC by-products characterisation
Flow cytometry
Simultaneous
expression of
markers
Limit of Detection
(LOD)
iPSC-derived cardiomyocytes: Cell impurities in cardiomyocytes
Limit of Quantification
(LOQ)

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IPSC by-products characterisation
QPCR
IPSC-derived cardiomyocytes: comparison of markers expression
Troponin
Myosin
Actinin

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IPSC by-products characterisation
Microscopy
IPSC-derived cardiomyocytes

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We know…we share:
Observations from our analytical experiments
Microscopy & QPCR
 Qualitative/Semi-quantitative methods
 Orthogonal methods for flow cytometry
 Microscopy: same antibody as for flow cytometry
but « pure »  availability !
 Supportive Data

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We know…we share:
Observations from our analytical experiments
Flow cytometry
ESC and IPSC a story a little bit different…
 Cells growing in colonies
 Spontaneous differentiation
 Problem of viability, cell recovery for some cell lines,…

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We know…we share:
Observations from our analytical experiments
Flow cytometry
SATISFACTORYFor identity testing
≥ 96 % ≥ 97 % 100 %
IPSC/ESC
≥ 93 % ≥ 95 % ≥ 96 %
IPSC derived
cardiomyocytes
Nanog Oct3/4 Sox2
Actinin Myosin Troponin

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We know…we share:
Observations from our analytical experiments
Flow cytometry
QUESTIONABLEFor impurities detection
Autofluorescence
LOQ (~0.22 %) ?

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Conclusions
PCR
 QPCR and microscopy: supportive data
 Flow cytometry: quantitative approach for identity tests
 Impurities detection: need for a new concept of test

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Conclusions
PCR
 QPCR and microscopy: supportive data
 Flow cytometry: quantitative approach for identity tests
 Impurities detection: need for a new concept of test
What is the next analytical step for cell manufacturing?
Chaparro & Linero. 2016. Advanced Techniques
in Bone Regeneration. Chapter 12.

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ACKNOWLEDGEMENTS
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Arnaud DELOBEL
Nathalie DRAUX
Estelle LARA
Capucine LEPERS
Tristan PRITCHARD-MEAKER
Nicolas THEYS
Sandra THYS
Marie TOUSSAINT
Fabian VANDERMEERS
Kassandra WATILLON

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valerie.deffontaine@quality-assistance.be
+32 71 53 47 81
www.quality-assistance.com
Technoparc de Thudinie, 2
B-6536 Donstiennes (Belgium)
Thank you for your attention
Any question?