Large Scale culture
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Mesenchymal Stem Cells
ESC/iPSC
CHARACTERIZATION
CD Surface Antigens
Chemokine Receptors
Cytokines
Mitogens
Immunological Features
SOURCE
Bone Marrow
Adipose Tissue
Umbilical Cord
Amniotic Fluid
Skeletal Muscle
& Others
ISOLATION
APPLICATIONS
Basic Biology
Cancer Biology
Genomics
Drug Discovery
Cell Therapy
EXPANSION
Multi-lineage Adult Stem Cells
(i.e. MAPC, MPLC, MIAMI, etc.)
MESODERM DIFFERENTIATION
TRANSDIFFERENTIATION
Neural
Hepatic
Endothelial
Tenocyte Chondrocyte Osteocyte
(tendon)
(cartilage)
(bone)
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Adipocyte
(fat)
Myoblasts
(muscle)
Human MSC Therapeutic Applications
Human MSCs have become of interest for clinical application due to:
• Capacity for homing and engraftment
• Wide-range differentiation potential
• Immunosuppressive attributes
Potential MSC Therapies:
• Graft versus Host Disease
• Crohn’s Disease
• Bone Defects/ Genetic Disease
• HSC Transplantation
• Cardiac repair
• NIH Clinical Trials search for mesenchymal stem (stromal) cells = >90 studies
(www.clinicaltrials.gov)
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Overview of MultiStem® Production Process
Lot Release & Product Characterization
Testing
Sterility
Potency
Identity and Viability
Stable Cytogenetics
Absence of tumorigenic potential in vivo
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MultiStem – Adherent Adult Stem Cell Platform-Athersys
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IND Protocol Title
Key elements
Phase I, Multicenter, Dose-Escalation Trial Evaluating
Maximum-Tolerated Dose of Single and Repeated
Administration of Allogeneic MultiStem® in Patients with
Acute Leukemia, Chronic Myeloid Leukemia, or
Myelodysplasia
IV-delivered product
Phase I: open label –
SAD, MAD
Adjunctive to BM/HSC
transplant
Phase I, Multicenter, Dose-Escalation Trial Evaluating the
Safety of Allogeneic AMI MultiStem® in Patients with
Acute Myocardial Infarction
Catheter-delivered
product
Phase I: open label (w/
registry)
Phase I, Multicenter, Dose-Escalation Trial Evaluating the
Safety of Allogeneic MultiStem® in Patients with Ischemic
Stroke
IV-delivered product
Phase I: blinded,
placebo controlled
Human MSC Therapeutic Applications
The processing of human MSCs for therapeutic application should be a
standardized GMP process, including defined:
(a) Starting material: donor, tissue origin, harvesting, enrichment method, etc.
(b) Tissue culture processing methods: culture medium, culture conditions, etc.
(c) Devices for cell culture: closed (or nearly closed) cell culture system
(d) Quality control:
> input material (donor) analysis
> active components or impurities
> expanded cell karyotype
Sensebé and Bourin Transplantation. 2009 May 15;87(9 Suppl):S49-53. Review.
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No inhibitor
Tyrphostin (IC50 20 M)
(PDGF inhibitor)
SB431542 hydrate
(IC50 10 M)
(TGFβR inhibitor)
SU 5402 (IC50 10-20
M)
(FGFR inhibitor)
10.38
9.76
4
6.92
8.03
20
5.05
5.38
40
1.01
0.71
2
7.21
3.12
10
6.35
3.46
20
2.88
2.29
2
10.77
8.28
10
4.09
2.82
20
3.46
3.31
10
9
8
7
6
5
4
3
2
1
0
15 pathways
Ng et al. Blood. 2008 Jul 15;112(2):295-307 2008
* Analysis of active pathways in hMSCs suggests that PDGF, TGFβ and FGF are
important signaling pathways for hMSC proliferation and differentiation
Wnt/β-catenin
Signaling
Wnt/βcatenin
Signaling
C14
PDGF
Signaling
C07
3
TGF-β
Signaling
PTEN
Signaling
PPAR
Signaling
PDGF
Signaling
p38 MAPK
Signaling
JAK/Stat
Signaling
Integrin
Signaling
4
Neuregulin
Signaling
Leukocyte
Extravasation
Signaling
JAK/Stat
Signaling
Integrin
Signaling
Insulin
Receptor
Signaling
Fold increase in viable cell number
IGF-1
Signaling
7 pathways
0
Huntington's
Disease
Signaling
0.5
GM-CSF
Signaling
1
ERK/MAPK
Signaling
O14
Actin
Cytoskeleton
Signaling
Cell Cycle:
G1/S
Checkpoint
Ephrin
Receptor
Signaling
1.5
-log(p-value)
3.5
ERK/MAPK
Signaling
p38 MAPK
Signaling
2
Ephrin
Receptor
Signaling
Day 7
Complement
and
Coagulation
2.5
EGF Signaling
Conc (M)
Interferon
Signaling
O07
Actin
Cytoskeleton
Signaling
Axonal
Guidance
Signaling
Cell Cycle:
G1/S
Checkpoint
Inhibitor
Leukocyte
Extravasation
Signaling
3
-log(p-value)
0
ERK/MAPK
Signaling
Death
Receptor
Signaling
Axonal
Guidance
Signaling
-log(p-value)
Active Signaling Pathways in Human MSCs
Osteogenesis
Chondrogenesis
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2
1
13 pathways
Day 14
Adipogenesis
A07
A14
StemPro MSC SFM
StemPro® MSC SFM Kit (Cat# A10332-01)
Consists of the following:
1.
StemPro® MSC SFM Basal Medium (1X)(500ml)
2.
StemPro® MSC SFM Supplement (6.67X)(75ml)
Note: Additional components required:
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•
CELLstart (XenoFree Substrate) (Cat# A1014201)
•
GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)
•
TrypLE Express (Cat# 12604013) (Animal Origin Free)
StemPro MSC SFM
Total Cell Expansion: STEMPRO MSC SFM
1800
9.0E+06
P = PDGF-BB
1400
Net Total Cell Number Per Flask
Relative Flourescence Units (RFU)
1600
B = bFGF
1200
T = TGFβ1
1000
800
600
400
8.0E+06
7.0E+06
6.0E+06
5.0E+06
4.0E+06
3.0E+06
2.0E+06
1.0E+06
0.0E+00
200
0
0
SCM
D+PBT
D
D+P
D+B
D+T
D+PB
D+PT
Growth Factor Supplementation
3
5
input cells = passage 5 human Bone Marrow MSCs (4-donor pool)
Adipocyte
(Oil Red O)
StemPro MSC SFM
Chondrocyte
(Alcian Blue)
Expansion
Differentiation
DMEM + 10% FBS
1
Passage
D+BT
Chondrocyte
(Toluidine Blue)
Beadchip Gene Array Analysis
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Chase et al. 2009 Submitted.
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DMEM + 10% MSC-Qualified FBS
STEMPRO MSC SFM
Osteoblast
(Alkaline Phosphase)
StemPro MSC SFM
Beta Test Data – Dr. Hideaki Kagami – The University of Tokyo
Primary Culture
in vivo ectopic bone assay
MEMalpha + 10% FBS
StemPro MSC SFM
Experimental Observations:
H&E staining; Data published in Agata et al. Biochem Biophys Res Commun 2009;382(2):353-8
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1)
Enhanced primary culture
efficacy (more cells faster)
2)
Lower alkaline phosphatase
activity in undifferentiated
cells
3)
Greater responsiveness to
osteogenic induction
4)
Confirmed in vivo ectopic
bone formation
StemPro MSC SFM
• A cGMP serum-free medium specially formulated for the growth and expansion of
human mesenchymal stem cells (MSCs).
PERFORMANCE - BETTER CELLS - CONVENIENCE
• Contains xenogenic components not amenable to most clinical applications
• Need for a second generation product: StemPro MSC SFM XenoFree
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StemPro MSC SFM XenoFree
StemPro MSC SFM XenoFree Kit (Cat# A11409SA)(Custom cGMP)
Consists of the following:
1.
StemPro® MSC SFM Basal Medium (Cat# A10334-01) (500ml)(1X)
2.
StemPro® MSC SFM Supplement (Cat# 09-0012SA) (5ml)(100X)
Note: Additional components required:
•
CELLstart (XenoFree Substrate) (Cat# A1014201)
•
GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)
•
TrypLE Express (Cat# 12604013) or TrypLE Select (Cat# 12563011) (Animal-Origin Free)
Complete cGMP XenoFree workflow: (1) Medium; (2) Substrate; (3) Harvest enzyme
For order inquiries please contact:

Sandy Kuligowski (sandra.kuligowski@lifetech.com) or

Lucas Chase (lucas.chase@lifetech.com)
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StemPro MSC SFM XenoFree: BM-MSC Expansion
DMEM + 10%
MSC-Qualified FBS
StemPro MSC SFM XenoFree: Net Population Doublings
(Human BM-MSC)
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Net Population Doublings
Passage 1
18
16
14
12
10
8
6
4
2
0
Passage 9
Expansion and Differentiation
StemPro MSC
SFM XenoFree
Set-up
1
2
3
4
5
6
7
8
9
Passage
DMEM + 10% MSC-Qualified FBS
StemPro MSC SFM XenoFree
StemPro MSC SFM XenoFree: Doubling Time
(Human BM-MSC)
Input cells = passage 5 human Bone Marrow MSCs (4-donor pool)
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Adipocyte
(Oil Red O)
Chondrocyte
(Alcian Blue)
Osteoblast
(Alkaline Phosphase)
Doubling Time (Hours)
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
Passage
DMEM + 10% MSC-Qualified FBS
Passage 5 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents
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StemPro MSC SFM XenoFree
CD34-Qdot ® 800
CD105-Alexa Fluor® 700
Negative markers-rPE
CD90-FITC
CD73-PerCP
Negative markers-rPE
Multiplex Flow Cytometry
StemPro MSC SFM XenoFree: BM-MSC Characterization
StemPro MSC SFM XenoFree
Passage 5
Passage 9
Marker
(% Positive)
(% Positive)
CD73+/NEG99.3
99.9
CD90+/NEG96.4
100.0
CD105+/NEG96.5
99.9
CD34+
0.1
0.6
DMEM + 10% MSC-Qualified FBS
Passage 5
Passage 9
Marker
(% Positive)
(% Positive)
CD73+/NEG99.7
100.0
CD90+/NEG97.9
100.0
CD105+/NEG98.1
100.0
CD34+
0.2
2.3
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
Jolene Bradford – Life Technolgies
Karyotype Analysis
CD90-FITC
Negative markers-rPE
Passage 5
Passage 9
Cells analyzed at Passage 5 and 9 = 46, XY= NORMAL KARYOTYPE
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Minimal Defining Criteria *:
(1)Adherence to plastic under standard
culture conditions (10% FBS-containing
medium)
(2) Characteristic Surface marker
expression
Positive (≥95%)
Negative (≤2%)
CD73
CD11b or CD14
CD90
CD34
CD105
CD45
CD79a or CD19
HLA-DR
(3) In vitro tri-lineage mesodermal
differentiation (osteoblasts,
chondrocytes, adipocytes)
*Dominici et al. 2006
CD73-PerCP
Multi-Parameter Human MSC Characterization
Flow cytometry combining
Click-iT® EdU proliferation analysis
WITH
Antibody-based immunophenotyping
Simultaneous testing for percentage proliferation with phenotype
characterization
Human
MSCs
0.5%
CD90-FITC
CD105-AlexaFluor® 700
Marker
99.5%
Fluorophore
CD73
PerCP
CD90
FITC
CD105
Alexa Fluor® 700
CD14
99.6%
0.4%
CD90-FITC
CD19
R-PE
Negative markers-rPE
CD45
HLA-DR
CD34
Cell proliferation
Click-iT EdU®
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Proliferating Human MSCs
detected with Click-iT® EdU reagents
Click-iT® EdU Alexa Fluor® 647 azide
FxCycle™ Violet stain
21.5%
FxCycle™ Violet DNA
CD34-Qdot® 800
Bright field
Hoechst
Click-iT® EdU
Click-iT® EdU
Alexa Fluor® 647 azide
DNA content
Qdot® 800
FxCycle™ Violet
DNA content
100%
CD90-FITC
100%
CD90-FITC
DMEM + 10% FBS - P3
NOTES:
• Input cells = P5 MSC (4-donor pool)
• P3 = 13 days in culture
• P9 = 42 days in culture
• Beadchip = HumanWG-6 v3.0
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R2 = 0.949
SP MSC SFM XF - P3
DMEM + 10% FBS – P9
R2 = 0.910
SP MSC SFM XF – P9
SP MSC SFM XF - P3
StemPro MSC SFM XenoFree: BM-MSC Characterization
R2 = 0.957
DMEM + 10% FBS – P3
StemPro MSC SFM XenoFree: BM-MSC Primary Isolation
StemPro MSC SFM XenoFree: Primary Expansion
(Human BM-MSC)
Net Cell Number Per Flask
1.2E+07
1.0E+07
8.0E+06
StemPro MSC SFM XenoFree - Primary Isolation
Passage 3
Marker
(% Positive)
CD73+/NEG99.8
+
CD90 /NEG
99.9
+
CD105 /NEG
99.7
CD34+
0.4
Human Serum
Removal
6.0E+06
4.0E+06
2.0E+06
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
0.0E+00
0
1
2
3
Passage
StemPro MSC SFM XenoFree
ADIPOCYTE
CHONDROCYTE
OSTEOBLAST
OSTEOBLAST
Alkaline Phosphase
(Day 14)
Alizarin Red S
(Day 21)
Differentiation (P3)
Expansion (P3)
StemPro MSC SFM
XenoFree
Oil Red O
(Day 14)
Alcian Blue
(Day 14)
Passage 3 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents
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StemPro MSC SFM XenoFree: ADSC Expansion
StemPro MSC SFM XenoFree: Net Population Doublings
(Human ADSC)
StemPro MSC SFM XenoFree: Doubling Time
(Human ADSC)
140
10
120
Doubling Time (Hours)
Net Population Doublings
9
8
7
6
5
4
3
2
100
80
60
40
20
1
0
0
Set-up
1
2
3
4
1
5
2
Passage
Control Medium
Control Medium
StemPro MSC SFM XenoFree
Human ADSC – SP MSC SFM XF
3
5
Adipocyte (Oil Red O)
Osteoblast (Alk Phos)
StemPro MSC SFM XenoFree
Chondrocyte (Alcian Blue)
Shayne Boucher – Life Technologies
NOTES:

• Input cells = passage 4 StemPro Human ADSCs

• Differentitaion = Passage 3, Day 14 – StemPro Differentiation Reagents
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4
Passage
StemPro MSC SFM XenoFree: Comparisons
Net Population Doubling: Competitor Audit
P3 StemPro MSC SFM
XenoFree
CD105-AlexaFluor® 700
Net Population Doublings
16
14
12
10
8
6
4
2
P3 Competitor X
CD105-AlexaFluor® 700
18
0
Set-up
1
2
3
4
5
6
7
Passage
10% MSC-Qualified FBS
CD90-FITC
StemPro MSC SFM XenoFree
CD90-FITC
Competitor X
Flow cytometry data generated by Jolene Bradford – Life Technologies
NOTE: input cells = passage 5 Human BM-MSCs (4-donor pool)

Chondrocyte
(Alcian Blue)
(Passage 3)
Expansion
(Passage 3)
StemPro MSC SFM XenoFree
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Competitor X
StemPro MSC SFM XenoFree
Passage 3
Marker
(% Positive)
CD73+/NEG98.8
CD90+/NEG100.0
CD105+/NEG100.0
+
CD34
0.3
Competitor X
Passage 3
Marker
(% Positive)
CD73+/NEG98.4
CD90+/NEG77.3
+
CD105 /NEG
100.0
CD34+
0.4
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
Cell therapy work flow
Isolate
Expand
Growth factors
& substrates
Qualify
Store
Monitor
ELISA
HLA typing
Cell selection
beads
Cryopreservation
media
Media, supplements
& single use
processing systems
Flow cytometry
Cell imaging
Processing
devices
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Dissociation
enzymes
Gene expression
Immune
response
monitoring
Closed System Xeno-Free MSC Isolation and Expansion
1.
2.
3.
Mononuclear cell
isolation using
the Sepax system
MSC expansion
using the
CLINIcell cassette
and StemPro
MSC SFM
XenoFree Medium
BM aspirate
4.
Autologous
MSC
product
MSC harvest
and wash
using TrypLE
Select and the
CytoMate Cell
Washer
The detailed MSC isolation and expansion workflow was employed to provide an autologous
MSC product from human bone marrow aspirate under completely closed conditions using
xeno-free cell culture reagents.
Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum
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5.
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Can achieve manufacturing runs equivalent to >200,000L of liquid media
Closed system considerations
Small Volume ”Pillow”-Universal Bags”
[5L, 10L & 20L]
New ‘9101’
PE Film
EVA Film
Large Volume Bags”
[100L ,200L, 500L & 1000L]
New ‘9101’
PE Film
 Pre-filled, stocked and ready when
you need them (reduced lead time)
 Easily customized to integrate with
automated systems and process
applications (connection methods, inline filtration, flow rates, tubing
lengths, re-circulation loops, etc.)
 Closed system, ideal for aseptic
processes/applications
 Conveniently designed for bench top to
larger-scale applications
 Media bag ONLY, not a bioreactor!
 Ideal for ‘daisy-chaining’ set-up
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Old
PE Film
(Stedim &
Crestbury)
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Bioreactor – Single use bioreactors
Connection Methods
•
•
•
•
Quick Connect (MPC)
SCD Tubing
Threaded Luers
UNIVERSAL BAGS!!!!!
Prefilled Wave O-Series Cellbag
• Ready to ship - media formulations include Sf-900II and Freestyle 293
• Convenient - Cellbag chambers are filled and ready to use
• Customizable - you choose the GIBCO medium formulation and volume
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Size
Working Volume
Cellbag10L/O
Cellbag20L/O
0.5L – 5L
1L – 10L
Processing using Dynal® ClinExVivo™ MPC
Sample bag
Sample collection
bag
Primary magnet
Cell depletion bag
Secondary magnet
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DYNAL® Magnetic Beads
Combining magnetic particles and single use cell
expansion technologies
Manufacturing validation of biologically functional T cells
targeted to CD19 antigen for autologous adoptive cell therapy.
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Hollyman D. et al. J Immunother. 2009
Feb-Mar;32(2):169-80.
Phase I Clinical Trial underway at Memorial Sloan Kettering
Dynabeads® SSEA-4
• Undifferentiated hESCs are efficiently depleted from the sample
• Higher purity of differentiated cells
• Differentiated cells remain unaffected after purification
Dynabeads® SSEA-4 are designed to remove
undifferentiated human embryonic stem cells from a
culture, providing you with highly pure and differentiated
stem cells for your translational applications
Differentiated Neural Stem Cells before and after depletion of SSEA-4+ cells
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SSEA-4 APC
SSEA-4 APC
14 %
0.2 %
esis
gen
eo
Ost
CD105
CD90
IgG1
BM MNC
Closed System Xeno-Free MSC Isolation and Expansion
Alkaline
Phosphatase
CD105
CD90
IgG1
Day 12 MSC
Osteogenesis
Von Kossa
(Calcium Deposit)
Chondrogenesis
IgG1
Alcian Blue
(Proteoglycan)
CD14
CD14/45
Day 12 Expanded MSCs
Adip
o
Day 12 expanded mononuclear cells reveals robust selection of CD90 and CD105-positive cells
in the absence of hematopoeitic antigen expression (CD14 and CD45), indicative of human
MSC expansion. Expanded cells reveal retained multipotency as shown by positive staining
results for osteogenesis, chondrogenesis and adipogenesis.
Total Cell Expansion: 3x105 BM MNCs
esis
Oil Red O
(Lipid Vessible)
2x107 MSCs
Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum
29
gen
Human MSC Culture Systems- Testing
Lot release- pathogen, sterility, identity
functional quality, viability, consistency
Efficacy tests- Karyotype, epigenome,
contaminating cells, HLA type, etc
30
Summary
• Therapeutic application of human MSCs will require standardized
GMP-compliant procedures and reagents
• We have developed two generations of cGMP-manufactured serumfree MSC expansion media, including StemPro MSC SFM XenoFree
• Expansion of cells in StemPro MSC SFM XenoFree provides retained
MSC phenotype and multipotency with a stable karyotype
• XenoFree medium applied to a closed isolation/expansion protocol
reveals a promising clinical culture system
• Continual collaboration will be critical to explore both in vitro and in
vivo applications
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Acknowledgements
Life Technologies:
Ruhr-Universität Bochum (Germany)
Uma Lakshmipathy
Shayne Boucher
Mohan Vemuri
Sandy Kuligowski
Doug Danner
Saki Phommachanh
Jean Donovan
Maureen Cook
Bob Kenderson
Alaine Maxwell
Kate Wagner
Mary Lynn Tilkins
Andy Campbell
Jolene Bradford
Scott Clarke
Christian Prante
Wolfgang Prohaska
Knut Kleesiek
A*STAR (Singapore)
Vivek Tanavde
Felicia Ng
Susie Koh
Konduru S. R. Sastry
The University of Tokyo
Hideaki Kagami
Hideki Agata
Case Western Reserve University
Luis Solchaga
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