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Generation of HLA universal platelets for regenerative applications
Constança Figueiredo
Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
IMGT/HLA database
12.672 HLA alleles
HLA loci are the most polymorphic of the entire human genome
A mean of 450 new alleles/ Year
Structure of HLA class I and II molecules
HLA-AHLA-B
HLA-DRHLA-DP
HLA Class I HLA Class II
HLA-BHLA-C
HLA-DPHLA-DQ
The function of HLA molecules
Antigen presentation
Efficient immune response against pathogens and cancer cells
�HLA Antigens = Major Antigens
�Non-HLA Antigens = Minor Antigens = Presented Peptides
HLA variability is the major hurdle for transplantation
MHC/non-MHC
MismatchImmune response
(humoral/cellular) Rejection
Standard Strategy to prevent rejection:Immunosuppression
Suppression of the recipient’s immune system
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.
Aim
1. develop tissues with low or no immunogenicity.
2. decrease the risk of HLA-mediated post-transplanttissue/organ failure.
Universal cells and tissues
Tissue(invisible)
CompetentImmune System
HLA TCR
(invisible)System
Alternative Strategy to prevent rejection:Reducing the organ‘s immunogenicity
Reducing the organ’s Immunogenicity
Gene silencing by RNA interference (RNAi):Small interfering RNA (siRNA)
Synthetic siRNA duplex
siRNA duplex
Cellular kinase
Andrew FireStanford University
Single-stranded siRNA in RNA-induced silencing complex (RISC)
Translational Block Mechanism Cleavage Mechanism
mRNA mRNA
Craig MelloUniversity of Massachusetts
Nobel Prize 2006
Silencing of β2-microglobulin in rat Lew fibroblasts
Figueiredo et al. BioMed Res Int, 2013
MHC silencing prolongs graft survival after allogeneic transplantation
Figueiredo et al. BioMed Res Int, 2013
Role of platelets
� Maintain Haemostasis
� Secretory functions
� Modulation of immune responses� Modulation of immune responses
� Regulate proliferation
Application of platelets in regenerative medicine
� Treatment of thrombocytopenia
� Wound healing
� Tissue remodelling� Tissue remodelling
� Drug carriers
Thrombocytopenia
Characteristics
• Low platelet number
• Major bleeding complications
HLA-universal platelets
Treatment optionsTreatment optionsTreatment optionsTreatment options
Ex-vivo induced megakaryocyte
progenitors
TPO receptor agonistsAllogeneic platelets
Alloimmunization against HLA class I
antigens
� Platelet
refractorinessdue to HLA antibodies
Treatment optionsTreatment optionsTreatment optionsTreatment options
In vitro production of Platelets to treat platelet transfusion refractoriness
Thrombopoiesis
Thrombopoietin (TPO)
Stem cell factor (SCF), IL-3IL-6, IL-11
Cell division Platelets
Endomitosis
MEP CFU-MK Promegakaryoblast Megakaryocyte
Proplatelet producing megakaryocyte
CD41 (GPIIb/IIIa)
CD42a (GPIX), CD42b (GPIb)
H1 shRNA EF1a GFP
Silencing HLA expression
T CA
A
GAG
5′- GAATGGAGAGAGAATTGAA
3′- TTCTTACCTCTCTCTTAACTT
T
A
HLA class I-specific silencing
Figueiredo et al, J mol Med, 2006
Figueiredo et al, Transfusion, 2007
Figueiredo et al, BioMed Res Int, 2013
Wiegmann & Figueiredo et al, Biomaterials, 2014
Polyploidy of differentiated megakaryocytes
PBMCs CD41+ shRNA_NS CD41+ shRNA_β2m
35% 33%0,1%
Ce
ll c
ou
nt
2n4n
8n
16n
PI HLA class I silencing HLA class I silencing HLA class I silencing HLA class I silencing
64%
shNS shβ2mNC
white bar = 50µm
shNS shβ2misotype
29.5 % 33.7 %0.0 %
ProPLTsProPLTsProPLTsProPLTs
PLTsPLTsPLTsPLTs
ProPLT/ PLT-production by differentiated MKs C
D42a
CD61
29.5 % 33.7 %0.0 %
Cell c
ou
nt
PAC-1
shNS + ADP shβ2m + ADPshNS
25.1% 29.4%2.0%
(n = 4)
Gras & Figueiredo et al. Hum Gen Ther, 2013
HLA-universal MKs protected from antibody-mediated cytotoxicity
sh
NS
NC PC aHLA03
shβ
2m
(n = 4, ***p<0.001)
HLA-universal MKs & PLTs prevent PLT refractoriness in a mouse model
NSG NSG
antiHLA ab
shNS shβ2mNC (ab only) ab + shNS ab + shβ2m
HLA-universal PLTs prevent PLT refractoriness in a mouse model
huC
D42a
shNS shβ2mIsotype control
Human PLTs in the circulation of NSG miceHuman PLTs in the circulation of NSG miceHuman PLTs in the circulation of NSG miceHuman PLTs in the circulation of NSG mice
huCD61
huC
D42a
NC (ab only) ab + shNS ab + shβ2m
(n = 3, *p<0.05, **p<0.01)
Gras & Figueiredo et al. Hum Gen Ther, 2013
Induced pluripotent Stem (iPS) cells as unilimited cell sources
Yamanaka S. Nature Medicine 2009.
Differentiation of iPSCs toward HLA-universal PLTs
iPSC colony Silencing HLA expression iPSC cultured in monolayer
Orbital shaker Megakaryocyte andPlatelet formation
BMP-4VEGF
TPO
Mesoderm
Megakaryocytes
Hematopoiesis/MK
SCFIL-3TPO
Xenofree conditions
Generation of HLA-universal iPSC lines
shNS shß2m
NC shβ2m
Co
un
ts
SSEA4
Tra-1-60Bef
ore T
d P1 P5
P10 P15
0
50
100
150shNS
shß2m
% H
LA
exp
ressio
n
Monolayer in Laminin
Co
un
ts
CD
42
a
isotype day 12
0.2 % 7.3 %
Generation of HLA-universal Megakaryocytes
MKs
CD
42
a
CD41
day 19 day 26
82.1 % 44.6 %
shN
S
shß2m
0
20
40
60
80
100
% H
LA
exp
ressio
n
Differentiation of Platelets
shβ2mNS
13.4 %18.6 %
CD
42a
CD61
CD
42a
Co
un
ts
Non-stimulated ADP + Thrombin
iPSC-derived Platelets respond to external stimuli
CD62P
MKsmembrane
PLTs
Large-scale production of platelets in bioreactors
CD
42
a
23.8 %
CD61
Summary
• The generation of in vitro pharmed genetically modified platelets isfeasible.
• IPSC serve as an unlimited cell source for the large-scale productionof platelets.
• Universal platelets bring the concept of Universal cells one stepcloser to reality.
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
Acknowledgements
Institute of Experimental Hematology, Hannover Medical School, HannoverThomas Moritz, Axel Schambach Nico LachmannMania Ackermann
LEBAO, Hannover Medical School, HannoverUlrich MartinStefanie WunderlichLena Engels
Stiftung für
Transfusionsmedizin
