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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 hematology Hôpital Saint Antoine Paris France

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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

HSCMyeloid HSC

CFU-GEMM

CFU-Meg

CFU-EBFU-E

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

MSCMS5MSC

MS5

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

in vivo Maturationof cReticulocytes

NOD-SCID mice

Day 18

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

2000x109 RBCs/packed RBC Unit

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

1 CD34+ 4 to 40x106 cRBC

1 CB 10-75 RBC units(5x106 CD34+)

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

Unlimited Source of Stem Cells:

Pluripotent Stem Cells

hESC & hiPSC

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

hiPSC hESC

Induction of Erythropoiesis and EB

hiPSC hESC

Characteristics of Erythroid Cells

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

Criteria for the Best Cell Sourcefor ex vivo RBC ProductionCurrent Opinion in Hematology, 2011

State of art

iPSCB HSC ES

40.106 RBC 5000 RBC 1000 RBC

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

Move forwardsfrom feasibility to effectiveness

Next Step : Scaling up for Industrial Production

Enjeu industriel de biotechnologie

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