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From stem cells to Red Blood Cells in vitro:
perspectives for transfusion medicine
Luc DOUAY
Saint Antoine research center
Department of hematologyHôpital Saint Antoine
ParisFrance
L.Douay & G.Andreu, Transfusion Medicine Review, April 2007
France
0%
10%
20%
30%
40%
50%
60%
70%
2010 2030 2050
Why Searching for New Sourcesof Blood Cells?
“Cell-Free hemoglobin-based blood substitutes and ris k of myocardial infarction and death :a meta-analysis”Natanson et al, JAMA April 2008
No substitute at date…
In vitro Generation of Human Blood Cells
Full differentiation
Functional cells
Massive in vitro production
From an unlimited stem cell source
A Perspective for Transfusion
HSCMyeloid HSC
lymphoid HSC
CFU-GEMM
CFU-GM
CFU-G
CFU-MCFU-Baso
CFU-Eo
CFU-Meg
CFU-EBFU-E
B Lymphocytes
T Lymphocytes
NK Cells
Dendritic cells
Dendritic cells
Neutrophiles
Mono/Macrophages
Basophiles
Eosinophiles
Platelets
Erythrocytes
104 CD34+ /mL
MS-5 or Hu-MSC
IL3
SCF
Epo
D0 D8 D11 D18
5ng/mL
3U/mL
100ng/mL
Proliferation and Erythroid Commitmenton in vitro Reconstituted Bone Marrow Microenvironment
CD34+ cells frombone marrow, blood, G-CSF leukapheresis, cord blood
Neildez et al, Nature Biotechnology 2002Giarratana et al, Nature Biotechnology, 2005
Reticulocytes
100
0
Cou
nts
1 10 102 103 104 1 10 102 103 104 1 10 102 103 104
99% 89% 23%
CD71GlycoA CD36
G6PD: 55 ± 6 UPK: 102 ± 9 U
Enzymes content
D18
Gel NuPAGE Tris-Acetate 3-8% Gel Tris-Glycine 10%
Les protéines membranaires des cRBCMembrane proteins
Functional Reticulocytes
Deformability of cRetStudied by Laser-assisted Optical Rotational Cell Analyzer
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0 5 10 15 20 25 30 35
Shear Stress (Pa)
Elo
ngat
ion
Inde
x (E
I)
native RBCs
native Ret
cRet
Hb%
Time (min.)
Total HbA, 96%Total HbF, 3.8%
F=
3.8
%
A1c
= 2
.4%
A2=
1.3%
%
20
15
10
5
0
0 1 2 3 4 5 6
A0
From Peripheral Blood From Cord Blood
Time (min.)
Total HbA, 28%Total Hb F, 72%
F
A1c
= 2
.4%
A0=
26%
Hemoglobin Content
Tonometric Oxygen Bindingof LK-cRet
Functional Hemoglobin
Fra
ctio
nals
atur
atio
n
Fra
ctio
nals
atur
atio
n
[2,3 DPG ]/Hb < 0.2 [2,3 DPG ]/Hb = 2-4
1.0
0.8
0.6
0.4
0.2
0.0
1 10 100 LOG (PO2, mm Hg)
[2,3 DPG]/Hb=2.4
[2,3 DPG]/Hb<0.2
Adult RBCLK-cRet
Fra
ctio
nals
atur
atio
n
Adult RBC
CB-cRet
Foetal RBC
PO2 (mm Hg)20 40 60 80 100 120 140
0.0
0.2
0.4
0.6
0.8
1.0
A RBC
CB-cRet
F RBCCB-cRetFoetal RBC
Equilibrium Oxygen Bindingof CB-cRet
CFSE
CD
71+
with
inC
FS
E+
cells
4.8%35.6% 18.8% 8.5%
Day 1 Day 2 Day 3 Day 5
Complete in vivo cRet Maturation
Day 1 Day 3 Day 5
LDS
CD34+
SCF+ FLT3-3, TPO+ IL3
D0 D7
HSC Expansion
D7 D25 D25 D32
Contact Contact
SCF+ Epo+ IL3 Epo
Erythroid differentiation
6
21
70
45
38
32
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9
1
10
100
1000
10000
100000
1000000
10000000
100000000
Enucleation rate (%)
Fold expansion
Days of culture
Enu
clea
tion
rate
(in %
of h
eov
eral
lpop
ulat
ion)
Total cellexpansion
(fold)
RBC from Cord Blood
0 7 11 15 18 22 25 29 32
Quantitative Estimations
Minimal Perspectives
Rare blood groups or complexe phenotypes
1% of transfused units
= 130,000 units/yr in USA
Quantitative Estimations
Minimal Perspectives
Rare blood groups or complexe phenotypes
1% of transfused units
= 130,000 units/yr in USA
Need for 3000 Cord Bloods each year(4.3 M birth potential)
A Banking Challenge
Undifferentiated Human ES Cells
Cell culture
SSEA-3 SSEA-4 TRA-1-60 TRA-1-81
Expression of membrane antigens
Nervous (Ect) System
Cartilage (M)
Glandular (E) Epithelium
Skin (Ect)
Muscle (M)
Respiratory (E)Epithelium
Formation of teratomasExpression of genes
Karyotype
Thomson et al., Science, 1998
bFGF + stroma
Adult/Fetal Fibroblasts
Gene transferby retro/lentiviral
infection
“ES-like” cells
Oct4/Sox2/C-Myc/Klf4
Takahashi et al., Cell 2006
Takahashi et al., Cell 2007Yu et al, Science 2007
Among 24candidate genes
Oct4/Sox2/Nanog/Lin28
Induced Pluripotent Stem Cells
BMP4FGFVEGFActivin A
Cell sorting CD34+
Erythroid differentiation protocol
BMP4, FLT3-L, SCF, IL3, EPO
Synthesis ofHb Emb. Fetal liver cell line
Erythropoiesis from hES in vitro
Bouhassira et al. 2005, 2006
Lanza et al. 2008
hES
hEB Blast colonies
30 to 60% cRBC
Synthesis ofHb Emb.
Hb F
HoxB4
IMR90-16 RBCs from hiPSC and hESC
2 hES cell lines: H1 and H9 (Thomson, Wicell)
Lapillonne et al, (2010), Haematologica
3 hiPS cell lines: 2 fetal (MRC5, IMR90) and 1 adult (FD-136)
Oct4
Sox2Nanog
Lin28
SCF, FLT3-l, TPO, BMP4, VEGF,IL-3, IL-6, Epo
hiPSC hESC
SCF, EpoSCF, Epo, IL-3
D0 D8 D28D11
Hu Plasma 20 days
D20 - hEB
Epo
First StepFormation of EB and induction of differentiation
Second StepDifferentiation / Maturation to cRBC
cRBCHu Plasma
1 – Mesodermal, hematopoietic and early erythroid cond itioning2 – No CD34+ selection3 – Erythroblast differentiation
Erythroid Differentiation
J0
J8
J11 J15
J20
60%
J28
hES
60% enucleation
20%
J0
J8
J11 J15
J20 J28
hiPSC
20% enucleation
β β β β αααα εεεε++++ΑγΑγΑγΑγ ζζζζ
5% 29% 43% 21.5% 1.5%
Identification of the Globin Chains - hES
GγγγγHeme
HPLC and mass spectrometry
Abs
orba
nce
220n
m (
mA
U)
Elution time (min)
HbF 80%
HbA 2.5%
Elution time (min)
% H
b
CO rebinding kinetics after flash photolysis
Functionality of the Hemoglobin - hESC
Abs
orba
nce
220n
m (
mA
U)
β β β β αααα ε +ε +ε +ε + ΑγΑγΑγΑγ ζζζζ
0% 29% 36% 22% 13%
Identification of the Globin Chains - hiPS
Gγ γ γ γ Heme
Elution time (min)
HPLC and mass spectrometry
% H
b
HbF 93%
HbA 2.5%
Elution time (min)
CO rebinding kinetics after flash photolysis
Functionality of the Hemoglobin - hiPSC
CB HSC ES Cells iPs Cells
Cont Cont Cont
•Donation dependant
•Logistic difficulties
•Batch production
•Limited and restrictedchoice of phenotypes :
1 phenotype/CB
•Rare source
•Ethical questions
•No choice of phenotype
•Low proliferation
•Enucleation •Low proliferation
•Enucleation
Pro Pro Pro
•Proliferation•Total maturation
•Unlimited source•Continuous production
•Unlimited source•Continous production•Choice of phenotype
Systems LU KEL P1
Antigens 1 2 3 4 5 1 2 1 2 1 2 1 2 3 4 1 3 1
1 - - - + + - + - + - + - + - + - - - 1,33E-04 226
2 + + - - + - + - + - + - + - + - - - 1,60E-04 272
3 + - + + - - + - + - + - + - + - - - 1,77E-05 30
4 - - - + + - + + - - + + - - + - - - 3,55E-05 60
5 + + - - + - + + - - + + - - + - - - 4,26E-05 72
6 + - + + - - + + - - + + - - + - - - 4,73E-06 8
7 - - - + + - + - + + - + - - + - - - 8,03E-05 136
8 + + - - + - + - + + - + - - + - - - 9,63E-05 164
9 + - + + - - + - + + - + - - + - - - 1,07E-05 18
10 - - - + + - + + - + - - + - + - - - 7,52E-05 128
11 + + - - + - + + - + - - + - + - - - 9,03E-05 153
12 + - + + - - + + - + - - + - + - - - 1,00E-05 17
13 - - - + + - + - + - + - + + - - - - 8,87E-06 15
14 + + - - + - + - + - + - + + - - - - 1,06E-05 18
15 + - + + - - + - + - + - + + - - - - 1,18E-06 2
16 - - - + + - + + - - + + - + - - - - 2,66E-05 45
17 + + - - + - + + - - + + - + - - - - 3,19E-05 54
18 + - + + - - + + - - + + - + - - - - 3,55E-06 6
19 - - - + + - + - + + - + - + - - - - 6,02E-05 102
20 + + - - + - + - + + - + - + - - - - 7,22E-05 123
21 + - + + - - + - + + - + - + - - - - 8,03E-06 14
22 - - - + + - + + - + - - + + - - - - 5,02E-06 9
23 + + - - + - + + - + - - + + - - - - 6,02E-06 10
24 + - + + - - + + - + - - + + - - - - 6,69E-07 1
FY
group O
ABORH KEL Prevalence in
the French population
Expected number of donors in
France
JK MNS
Antigens combinations theoretically useful to manageallo-immunized patients
Th.Peyrard et al, Transf Med Rev. 2011 Mar 3
10 selected iPS cell line could provide compatible RB Cto these alloimmunized patients
Systems LU KEL P1
Antigens 1 2 3 4 5 1 2 1 2 1 2 1 2 3 4 1 3 1
1 - - - + + - + - + - + - + - + - - - 1,33E-04 226
2 + + - - + - + - + - + - + - + - - - 1,60E-04 272
4 - - - + + - + + - - + + - - + - - - 3,55E-05 60
7 - - - + + - + - + + - + - - + - - - 8,03E-05 136
8 + + - - + - + - + + - + - - + - - - 9,63E-05 164
10 - - - + + - + + - + - - + - + - - - 7,52E-05 128
12 + - + + - - + + - + - - + - + - - - 1,00E-05 17
17 + + - - + - + + - - + + - + - - - - 3,19E-05 54
18 + - + + - - + + - - + + - + - - - - 3,55E-06 6
20 + + - - + - + - + + - + - + - - - - 7,22E-05 123
Expected number of donors in
FranceABO
RH KEL FY
group O
JK MNS Prevalence in the French population
Th.Peyrard et al, Transf Med Rev. 2011 Mar 3
Comparison to a cohort of 16,486 consecutive alloim munized patients
Systems LU KEL P1
Antigens 1 2 3 4 5 1 2 1 2 1 2 1 2 3 4 1 3 1
1 - - - + + - + - + - + - + - + - - - 1,33E-04 226
2 + + - - + - + - + - + - + - + - - - 1,60E-04 272
4 - - - + + - + + - - + + - - + - - - 3,55E-05 60
7 - - - + + - + - + + - + - - + - - - 8,03E-05 136
8 + + - - + - + - + + - + - - + - - - 9,63E-05 164
10 - - - + + - + + - + - - + - + - - - 7,52E-05 128
12 + - + + - - + + - + - - + - + - - - 1,00E-05 17
17 + + - - + - + + - - + + - + - - - - 3,19E-05 54
18 + - + + - - + + - - + + - + - - - - 3,55E-06 6
20 + + - - + - + - + + - + - + - - - - 7,22E-05 123
Expected number of donors in
FranceABO
RH KEL FY
group O
JK MNS Prevalence in the French population
Th.Peyrard et al, Transf Med Rev. 2011 Mar 3
Search for these potential donorsin the french blood donor registry (1.7M)
98,67
99,1399,28 99,35 99,39 99,41 99,42 99,42
99,71100,00
95,53
99,43
95
96
97
98
99
100
hiPSC1
hiPSC8
hiPSC10
hiPSC2
hiPSC18
hiPSC17
hiPSC7
hiPSC12
hiPSC4
hiPSC20
A1 publicneg
3 “universal” iPS cell linesto provide compatible RBC
to 99% alloimmunized patients
iPS banking for transfusion purpose
Th.Peyrard et al, Transf Med Rev. 2011 Mar 3
Crucial points to be solved for iPS clinical useCurrent Opinion in Hematology, 2011
Defining GMP conditions for industrial production
International Consortia
• France (EFS) iPS « Stemred »
• US (DARPA) CB « Blood Pharming »
• GB (MRC) ES « Redontap »
Institut deGénétique et deBiologie Moléculaire et Cellulaire, Illkirch
Stéphane VivillePhilippeTropel
Université Pierre et Marie Curie, Paris
Daniela BoehmIsabel DornSabine FrançoisMarie-Catherine GiarratanaLaurence HarmandNicolas HébertSéverine JollyLadan KobariHélène Lapillonne Tiffany MarieChristelle Mazurier
Luc Douay
INSERM U46Henri Wajcman
INSERM U473M.C. MardenL. KigerTh.Cynober
CNRGSPY Le PennecTh.Peyrard
Unité de Pathologie Moléculaire du Globule Rouge,, Hôpital Edouard Herriot, Lyon
Alain Francina
EFS Ile de France
Hélène Rouard
INTSJP.CartronG.AndreuL.Bardiaux
Institut Pasteur
I.SafeukuiP.David
A. BennaceurF.Yates
INSERM U802