Stem cell Research in Friedreich Ataxia

Hélène Puccio Research Director Inserm [email protected]

Translational Medicine and Neurogenetics

Fundamental and pathophysiological mechanisms in recessive ataxia Friedreich ataxia (Frataxin)

Fe-S cluster metabolism

X-linked sideroblastic anemia with ataxia (ABCb7)

CoQ10 metabolism

Autosomal Recessive Cerebellar Ataxia type 2 (ADCK3)

FUNCTION

THERAPY

FRIEDREICH ATAXIA FRATAXIN

PHYSIOPATHOLOGY Knockout Knockdown

Cellular Models

Mouse Models

Cardiac Neuronal Muscular Hepatic

What is a Stem Cell? stem  cell SELF-­‐RENEWAL (copying)

stem  cell

DIFFERENTIATION (specializing)

specialized  cell e.g.  muscle  cell,  nerve  cell

A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues

Why are stem cells interesting? They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses => Regenerative medicine: too early, not scientifically mature They can replicate themselves over and over for a very long time. Understanding how stem cells develop into healthy and diseased cells will assist the search for cures. Drugs can be screened by testing them on human embryonic stem cells or the specialized cell derived from them.

Stem Cell Jargon Potency

A measure of how many types of specialized cell a stem cell can make

Pluripotent Can make all types of specialized cells in the body

Multipotent Can make multiple types of specialized cells, but not all types

Where do we find stem cells?

Mul=potent

embryonic  stem  cells blastocyst  -­‐  a  very  early embryo Pluripotent/To=potent

Assue  stem  cells

fetus,  baby  and  throughout  life

Induced pluripotent stem cells (iPSC) iPS are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, i.e. an adult skin cell • iPS were first produced in 2006 from mouse cells and in 2007 from human cells. • iPS are not totipotent and do not involve the destruction of an embryo.

Induced pluripotent stem cells (iPSC) ‘gene=c  reprogramming’ =  add  certain  genes  to  the  cell cell  from  the  body

induced  pluripotent  stem  (iPS)  cell behaves  like  an  embryonic  stem  cell

differen=a=on culture  iPS  cells  in  the  lab

Advantage:  no  need  for  embryos!

all  possible  types  of specialized  cells

Stem Cell: Challenges

A s n o iti d n o rc e d n u w o r g grow under c

stem  cells

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onditions B

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skin

neurons

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blood di t i on

sD

liver

Friedreich ataxia (FRDA) Most common recessive ataxia (1/30 000) progresssive mixed spinocerebellar and sensory ataxia hypertrophic cardiomyopathy increased incidence of diabetes

Frataxin

(GAA) 7-34

Normal (GAA)100-1000

FA

Transcriptional silencing

Frataxin

Frataxin is a mitochondrial protein Ubiquitously expressed (expressed in all tissues) Exact function of frataxin is unclear and very controversial closely linked to iron homeostasis mitochondrial function (energy production)

Why do we need new models for FRDA? Available cells for patients (fibroblasts and lymphocytes) - useful for some studies BUT are not the affected tissues - neurons, cardiomyocytes, beta-cell from pancreas Autopsy material is important to understand disease BUT late stages Mouse models developed - complete absence of frataxin: very useful for pathophysiology BUT more severe - GAA knockin or transgenic: very useful for looking mechanism of gene silencing by GAA and testing compounds BUT mildly affected

WE WOULD LIKE a human model, with large GAA repeats and low frataxin of affected tissues to be able to confirm hypothesis uncovered in current models and test new hypothesis.

Development of a GAA-based model induced  Pluripotent Stem  cell  (iPS) Somatic cells (fibroblasts)

Reprogrammed 4 fibroblast lines 1. Control female 25 yrs 2. Control male 50 yrs 3. FA female GAA= 600/830 4. FA female GAA= 430/900

feeders

iPS colonie

Reprogramming (pluripotency factors) Oct4, Sox2, Nanog, Lin28

iPSC

Differentiation Specific cell types

neurons, cardiomyocytes, … = genetic information than the starting somatic cell

Collaboration Stéphane Viville

Marie Wattenhofer

Bonafide iPS- are the iPS pluripotent

1.  Expression  of  endogenous  ES  markers 2.  Shutdown  of  len=virus  factors 3.  Normal  caryotype 4.  Embryoid  Bodies  (3  Germline  layers) 5.  Teratoma  forma=on:   -­‐  Ectoderm-­‐derived -­‐  Endoderm-­‐derived -­‐  Mesoderm-­‐derived

neural  Assue

Gut-­‐like epithelium

neural  ganglion

Epidermis

carAlage

Marie Wattenhofer

3000 bp

500 bp

1000 bp

FRDA iPS cells retain pathological GAA size and low levels of frataxin 500 bp

CT136 4L

FXN mRNA levels reduced

B

100

iPSCs FD141 4L

45 46 47 48 49 50

M FD141 43

28 29 30

FD135 4L

C 51 54 55 56 57 58mRNA level normalised FXN 60 61 62

M FD135 28 29 30 32 33 FD136 21 22 23

GAA expansion of pathological size

80

Tra1-60

Tra1-81 hFXN 1-210

CT136-4L clone 34

60

000 bp

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000 bp

3000 bp

500 bp

1000 bp

CT -4L (n=5)

1000 bp

CT136-4L 38 CT -4L cloneFD135-4L (n=5)

clones (n=4)

CT136-4L CT145-4L FD141-4L FD135-4L

FD135-4L clone 30

500 bp

control

hFXN 42-210

20

FA1

FXN protein levels reduced cl 34

cl 25

cl 2

cl 51 cl 49

FD141-4L clones (n=4)

hFXN 81-210

ß-tubulin

cl 30

hFXN 1-210

FD141-4L clone 49 hFXN 42-210 FD135-4L clones (n=4)

FD141-4L clones (n=4)

hFXN 81-210

FD141-4L clone 51

ß-tubulin

Marie Wattenhofer and Aurore Hick

Differentiation of iPS cells into neuronal and cardiac lineage

1

2

3

4

Mesoderm iPS cells

pleas

Generation of neural cell from iPS (Control and FRDA)

dNS FD135-4L

C M

Please choose theraph g you prefer Frataxin mRNA level (NS CT136 4L clone 38 set at 100 %)

NS N

i

N

NS

35-4L FD141-4L e 30 clone 51

dNS FD141-4L

120 100 80 60 40 20 0

Frataxin mRNA level (iPSC CT136 4L clone 25 set at 100 %)

dNS CT145-4L

120 100 80 60 40 20 0

CT FRDA NS NS

CT

FRDA

iPSC

CT F

NS

of D1, D2 and D3, o star t t with control. Could you please add the c Collaboration Massimo Pandolfo and Satyan Chintawar

D2

C

D3

Generation of neural cell from iPS (Control and FRDA)

Collaboration Massimo Pandolfo and Satyan Chintawar

30

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FRDA neurons are functionnally impaired

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t e xt

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CT 136 n =15

CT 145 n = 13

F D141

F D145

F A 13 5 F A 1 41 C T136- 2C0 n=16 7 n =24 7 n =24 5 n

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mitochondrial membrane potential CT145-4L

FD135-4L

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Electrophysiological studies

%of cells

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FA 1 3 5 n= 15 F D135

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FA 1 4 1 n =15

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FD135-4L

Collaboration Massimo Pandolfo and Satyan Chintawar

E2 T M R M M e a n fl u o re s c e n c e i n te n s i ty

50

TM R M M e a n f l u o r e sc e n c e i n t en s i ty

u rre ntn t NN aa CCu rre EEvo vo ke ked d AA P P p onnt an eo us A P SSpo t. AP

60

% o f ce l l s p a r ti c i p a ti n g in s p o n ta n e o u s c a l c i u m o sc i l a ti o n s

D5 60

**

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F A 1 35 F A 14 1 C T 136 C T** 1 45 n=24 5 n=20 6 n=16 7 n=24 7

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F A 135 F A 141 C T 136 C T 145

F F A 135 F A 141 C T 136 C T145

90pA

2m0VmV

0

20mV

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% o f c e l l s p ar ti c i p s p o n t an e o u s c a l ci u

CT

%o

CT

- 80

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Differentiation of iPS cells into cardiomyocytes

iPS

EB

Cardiogenic factors Beating cardiomyocytes

Control

Aurore Hick

hFXN/GAPDH

FRDA cardiomyocyte present a mitochondrial defect

*

** * p Promising for understand the step leading to cell dysfunction, for screening possible therapeutic compounds

Perspectives: stem cell in FA Several different labs (at least 8) have generated iPS cells for FA - neurons, cardiomyocytes (efforts in making pancreatic cells) - pathophysiological studies - screen candidate drug compounds - large scale unbiased screen

Several laboratories (Australia and UK) working on regenerative medecine - Mesenchemal Stem Cell (bone-marrow/blood cord) in mouse - iPS cell injection in mouse and rat CAREFULL: TOO EARLY TO THINK ABOUT THERAPEUTIC APPLICATION

FRDA and Fe-S biosynthesis

Collaborators

Brahim Belbellaa Lena Beilschmidt Florent Colin Aurore Hick Alain Martelli Morgane Perdomini Laurence Reutenauer Nadège Vaucamps

Sandrine Ollagnier de Choudens (CEA-Grenoble) Yvain Nicolet/Juan Fontecilla (IBIS-Grenoble) Cécile Bouton/JC Drapier (CNRS-Gif-sur-Yvette) Arnold Munnich / Agnès Rötig (Necker-Paris) Christine Tranchant (CHU Strasbourg) Massimo Pandolfo/Satyan Chintawar (Hôpital ULB- Belgium) Hervé Puy (Centre Français des Porphyrie- Paris) Matthias Hentze / Bruno Galy (EMBL-Heidelberg) Christopher Pearson (Hosp.Sick Children, Toronto) Stéphane Viville (IGBMC) Michel Koenig (IGBMC)

Nadège Carelle-Calmels Stéphane Schmucker Marie Wattenhofer-Donzé ARCA2 and CoQ10 biosynthesis Floriana Licitra Leila Laredj CAG repeat instability Karine Merienne Claudia Andretta Agathi Goula