generation of hla universal platelets for regenerative applications constança figueiredo institute...
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Generation of HLA universal platelets for regenerative applications
Constança Figueiredo
Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
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IMGT/HLA database
12.672 HLA alleles
A mean of 450 new alleles/ Year
HLA loci are the most polymorphic of the entire human genome
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Structure of HLA class I and II molecules
HLA-AHLA-BHLA-C
HLA-DRHLA-DPHLA-DQ
HLA Class I HLA Class II
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The function of HLA molecules
Antigen presentation
Efficient immune response against pathogens and cancer cells
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HLA Antigens = Major Antigens
Non-HLA Antigens = Minor Antigens = Presented Peptides
HLA variability is the major hurdle for transplantation
MHC/non-MHCMismatch
Immune response(humoral/cellular)
Rejection
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Suppression of the recipient’s immune system
Standard Strategy to prevent rejection:Immunosuppression
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Control HLA expression in a allele-, gene- or class-specific way.
In a clinical setting, this would allow to
1. develop tissues with low or no immunogenicity.
2. decrease the risk of HLA-mediated post-transplanttissue/organ failure.
Aim
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Universal cells and tissues
HLA TCR
Tissue(invisible)
CompetentImmune System
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Reducing the organ’s Immunogenicity
Alternative Strategy to prevent rejection:Reducing the organ‘s immunogenicity
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Gene silencing by RNA interference (RNAi):Small interfering RNA (siRNA)
Synthetic siRNA duplex
siRNA duplex
Cellular kinase
Single-stranded siRNA in RNA-induced silencing complex (RISC)
Translational Block Mechanism Cleavage Mechanism
mRNA mRNA
Andrew FireStanford University
Craig MelloUniversity of Massachusetts
Nobel Prize 2006
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Silencing of β2-microglobulin in rat Lew fibroblasts
Figueiredo et al. BioMed Res Int, 2013
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MHC silencing prolongs graft survival after allogeneic transplantation
Figueiredo et al. BioMed Res Int, 2013
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Role of platelets
Maintain Haemostasis
Secretory functions
Modulation of immune responses
Regulate proliferation
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Application of platelets in regenerative medicine
Treatment of thrombocytopenia
Wound healing
Tissue remodelling
Drug carriers
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Thrombocytopenia
Characteristics
• Low platelet number
• Major bleeding complications
Ex-vivo induced megakaryocyte
progenitors
TPO receptor agonistsAllogeneic platelets
Alloimmunization against HLA class I
antigens
Platelet refractoriness
due to HLA antibodies
HLA-universal platelets
Treatment options
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In vitro production of Platelets to treat platelet transfusion refractoriness
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Thrombopoiesis
MEP
Thrombopoietin (TPO)
Stem cell factor (SCF), IL-3IL-6, IL-11
CFU-MK Promegakaryoblast
Cell division
Megakaryocyte
Proplatelet producing megakaryocyte
Platelets
CD41 (GPIIb/IIIa)
CD42a (GPIX), CD42b (GPIb)
Endomitosis
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T CAAGAG
5′- GAATGGAGAGAGAATTGAA3′- TTCTTACCTCTCTCTTAACTT
T
A
HLA class I-specific silencing
H1 shRNA EF1a GFP
Silencing HLA expression
Figueiredo et al, J mol Med, 2006Figueiredo et al, Transfusion, 2007Figueiredo et al, BioMed Res Int, 2013Wiegmann & Figueiredo et al, Biomaterials, 2014
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Polyploidy of differentiated megakaryocytes
PBMCs CD41+ shRNA_NS CD41+ shRNA_β2m
35% 33%0,1%
Cel
l co
un
t
PI
2n4n
8n
16n
HLA class I silencing
64%
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shNS shβ2mNC
white bar = 50μm
CD
42a
shNS shβ2m
CD61
isotype
29.5 % 33.7 %0.0 %
ProPLTs
PLTs
Cel
l co
un
t
PAC-1
shNS + ADP shβ2m + ADP shNS
25.1% 29.4%2.0%
(n = 4)
Gras & Figueiredo et al. Hum Gen Ther, 2013
ProPLT/ PLT-production by differentiated MKs
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HLA-universal MKs protected from antibody-mediated cytotoxicity
sh
β2
ms
hN
S
NC PC aHLA03
(n = 4, ***p<0.001)
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HLA-universal MKs & PLTs prevent PLT refractoriness in a mouse model
NSG NSG
antiHLA ab
shNS shβ2mNC (ab only) ab + shNS ab + shβ2m
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HLA-universal PLTs prevent PLT refractoriness in a mouse model
huCD61
huC
D42
a
shNS shβ2mIsotype control
NC (ab only) ab + shNS ab + shβ2m
Human PLTs in the circulation of NSG mice
(n = 3, *p<0.05, **p<0.01)
Gras & Figueiredo et al. Hum Gen Ther, 2013
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Induced pluripotent Stem (iPS) cells as unilimited cell sources
Yamanaka S. Nature Medicine 2009.
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Differentiation of iPSCs toward HLA-universal PLTs
iPSC colony Silencing HLA expression iPSC cultured in monolayer
Orbital shaker Megakaryocyte andPlatelet formation
BMP-4 VEGF
TPO
Mesoderm
Megakaryocytes
Hematopoiesis/MK
SCFIL-3TPO
Xenofree conditions
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Generation of HLA-universal iPSC lines
SSEA4
Tra-1-60Before
Td P1 P5
P10 P150
50
100
150shNSshß2m
% H
LA
exp
ress
ion
shNS shß2m
Monolayer in Laminin
NC shβ2m
Co
un
tsC
ou
nts
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CD41
isotype day 12
day 19 day 26
0.2 % 7.3 %
82.1 % 44.6 %
Generation of HLA-universal Megakaryocytes
shNS
shß2m
0
20
40
60
80
100
% H
LA
exp
ress
ion
MKs
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Differentiation of Platelets
CD61
shβ2mNS
13.4 %18.6 %
CD
42a
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CD62P
Co
un
tsNon-stimulated ADP + Thrombin
iPSC-derived Platelets respond to external stimuli
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MKsmembrane
PLTs
23.8 %
Large-scale production of platelets in bioreactors
CD61
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Summary
• The generation of in vitro pharmed genetically modified platelets is feasible.
• IPSC serve as an unlimited cell source for the large-scale production of platelets.
• Universal platelets bring the concept of Universal cells one step closer to reality.
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Institute for Transfusion Medicine, Hannover Medical School, Germany.Rainer Blasczyk Dominca RatusznyLaura Schlahsa Haijiao ZhangAnn-Kathrin Börger Stefanie VahlsingLilia Goudeva
Helmholtz Center for Infection Research, Braunschweig, GermanyCarlos GuzmanKai Schulze
Institute of Experimental Hematology, Hannover Medical School, HannoverThomas Moritz, Axel Schambach Nico LachmannMania Ackermann
LEBAO, Hannover Medical School, HannoverUlrich MartinStefanie WunderlichLena Engels
Acknowledgements
Stiftung für Transfusionsmedizin