Mariana Laura Casalia
Laboratorio de Terapias regenerativas y protectoras del Sistema Nervioso
Fundación Instituto Leloir
Facultad de Farmacia y Bioquímica-UBA
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Epilepsy is the third most common neurological disorder,
affecting patients of all ages.
It is estimated that 1% of the global population will develop
epilepsy at some point in their lives.
The prevalence in Argentina ranges from 4 to 10 per 1000
habitants.
It is caused by an uncontrolled and hyper-synchronized
electrical activity.
It is usually diagnosed after the patient suffers 2 crisis of
unknown cause.
The available therapies act on the symptoms and not the
causes.
The field is in need of new models for this disorder, which
would allow an advancement in the knowledge of the
pathology and may provide new treatments.
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The Benign Focal Childhood Epilepsies (BFCEs) represent the
most prevalent syndromes in the pediatric population.
In most cases the phenotype is reversed in the second
decade of life.
Mutations in the FGD6 gene (FYVE, RhoGEF and PH domaincontaining protein 6) in patients with BFCEs could be a
possible genetic cause.
Genomic Epilepsy: EXOME SEQUENCING
Kauffman1,2
de
Silva3;
Morón1;
; Dolores González
Damián
1
2,3
Marta Córdoba ; Silvia Kochen
Marcelo Andrés
Nahuel Pereira
Consalvo3;
Síndrome de
Panayiotopoulos
Epilepsia no clasificada
GA
19
18
17
Criterios incompletos
para Síndrome de Panayiotopoulos
GA
12
10
4
18m
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The reprograming and subsequent differentiation of
patient-derived cells allow to generate a cellular model
of the disease.
The phenotype observed in BFCEs could be due to a
disruption at the synaptic circuits level.
Generate a model of the pathology by reprogramming
and differentiation of fibroblasts derived from donors
(patients and controls)
Identify molecular and functional alterations in those
cells.
Contribute to the knowledge of the pathophysiology of
the epilepsy with possible genetic etiology
Introduction

human
stem cell
obtained
fibroblats
genes.
induced
pluripotent
clones (hIPS) can be
by transducing adult
with 4 reprogramming
Skin biopsy
Derivation of fibroblast
Reprograming
Characterization and
clone selection
Differentiation into
neuroepithelial tissue
Characterization of the NE
Functional assays of the NE
Epilepsy model
Results
Fibroblas cell lines derived from donors
Sample type
Type
F5
Control
Non related
F6
Control
Non related
F9
patient
FGD6 mutation in homozygosis
F12
Control
FGD6 mutation in heterozygosis
F13
patient
FGD6 mutation in homozygosis
F14
Control
FGD6 mutation in heterozygosis
•
•
All donors sign an informed
consent
We work under the approval
3mm committee of
of the bioethical
FIL and Ramos Mejia Hospital
Results
Dermis/epidermis
dermis
grasa/dermis
F5
F5
F5
F5
Day19 - 40X
Day 12 - 40X
Day 9 - 5XDay 9 - 40X
Pasaje 1 (1:1)
Dermis-epidermis
Dermis-epidermis
Dermis-epidermis
Results
Time line of the reprograming protocol
FB medium
D-1
D0
hIPS medium
D2
D6
Plate fibroblast Transfect the STEMCCA
Plate on MEF
Fibroblast
D30
Pick clones
hIPS clone
Gustavo Mostoslavsky
Results
Mycoplasma detection by PCR
Transgene insertion by PCR
1 2 3 4 5 6 7 8 9 10 11 12 13
Expression of the RNA with
the mutation of interest
Karyotyping
Pluripotency
by RT-PCR
Silencing by RT-PCT
Pluripotency by ICQ
dapi
Nanog
Tra 1-80
dapi
Sox2
Oct4
Results
Spontaneous differentiation
Day 0
Day 4
Day 7
Make EBs
RNA
sample
Plate EBs
EB day 4
EB day 10
A
B
Day 20
ICQ sapmple
EB day 20 – Beating
Results
Spontaneous differentiation
by ICQ
dapi
by qRT-PCR
Dcx
Tuj
AFP
Gata4
troponina
desmina
ectoderm
dapi
endoderm
dapi
mesoderm
endoderm
mesoderm
ectoderm
Results
Take sample
D0
D4
Suspension
½ hIPS s/fgf
D7/8
D25
D17
Suspension
Suspension
adherent
NDM + GDNF + BDNF
NIM
NIM
NIM
+ IGF1+ cAMP + Asc. Acid
4W
Make
EBs
Change to
½ NIM
Plate onto
a 6w plate
Isolate and
expand
NE cells
8W
Plate
NE to
coverslips
Include into
Matrigel drops
hIPS
EBs
NE
NE
NE
H. Zhang 2010. Differentiation of Neural Precursors and Dopaminergic Neurons from Human Embryonic Stem Cells.
Lancaster 2013. Cerebral organoids model human brain development and microcephaly
NE
10W
The NE presents diverse neuronal subtypes and
structures reminiscent to neural tissue
LHX2
Pax6
GAD67
BIII tub
dapi
dapi
suspension
Otx2
Dcx
FoxA2
adhesion
TH
dapi
adhesion
adhesion
Hematoxylin eosin
Foxa2
Calb
Quantification in progress
dapi
suspension
Dlx2
suspension
Masson
trichrome
Preliminary Results
78% of the cells in patients and controls samples are BIIItub +
TUJ
control
paciente
100
50
10
se
m
0
4s
em
% celulas BIII Tub+/Dapi+
150
Preliminary Results
Patient’s NE expresses immature neuronal markers for longer periods
OTX2 BIIItub Dapi
control
pacient
50
control
pacient
e
150
% celulas PAX6+/Dapi+
*
*
100
50
PAX6
10
se
m
10
se
m
*p<0,004 Multiple t-test con correccion para comparaciones
multiples del metodo de Holm Sidak
4s
em
0
0
4s
em
OTX2
% celulas Otx2+ / Dapi+
150
100
PAX6 BIIItub Dapi
PAX6
OTX2
*p<0,00001 Multiple t-test con correccion para comparaciones
multiples del metodo de Holm Sidak
Preliminary Results
Neurons are electrophysiologically active with Na+ and Ca++ functional
channels
Electrophysiology – Patch clamp
NE of 12 weeks of differentiation
4 weeks of differentiation
INa + ICa
8 weeks of differentiation
Protocol
Artifact
Protocol
Artifact
ICa
No ICa
TTX 10uM
Cadmium 100uM
Preliminary Results
Patient’s neurons lack define neuronal processes at 7 weeks
CONTROL
PATIENT
TUJ
control
paciente
% celulas BIII Tub+/Dapi+
150
100
50
TUJ (BIII TUB) Dapi
Undefined process
10
se
m
Defined process
4s
em
0
Preliminary Results
Smaller axonal length in BIIItub(+) cells derived from patients
Axonal length
(um)
Axonal length in BIII tub/Tau-1
positive cells
CONTROL
PATIENT
**
CONTROL
PATIENT
P 0.0064, t-test. Axonal lenght in Tuj+Tau-1 positive
cells in an NE
Axón
Axón
Tuj (BIIITub) Tau-1 Dapi
R.Quinta
Preliminary Results
Absence of Syn-1 clusters in Patient’s NE samples
CONTROL
clusters
PATIENT
SYN-1 TUJ (BIII TUB) Dapi
No clusters
R.Quinta
Summarize
 The NE presents diverse neuronal subtypes and structures reminiscent
to neural tissue
 There are no differences between the amount of BIIItub + cells in
patients and control NE samples
 Patient’s NE expresses immature neuronal markers for longer periods
 Neurons are electrophysiologically active with Na+ and Ca++
functional channels
 Patient’s neurons lack defined neuronal processes at 7 weeks of
differentiation
 Smaller axonal length in BIIItub + cells derived from patients were
found
 Absence of Syn-1 clusters in Patient’s NE samples were observed
 Possible delay in the maturation of the patient-derived neurons
• Further characterization in progress
✔
Skin biopsy
✔
Derivation of fibroblast
✔
Reprograming
✔
Characterization and
clone selection
✔
Differentiation into
neuroepithelial tissue
⏏ ⏏
Characterization of the NE
Functional assays of the NE
Epilepsy model
Verónica Cavaliere Candedo
Juan Cruz Casabona
Isabel Farias
Joaquín V. Gonzaáez
Carina Ferrari
Fernando Pitossi
Marcelo Kauffman –Neurology Service- Ramos Mejia Hospital
Juana Pasquini- Laboratory of Molecular Neuropharmacology- FMED- UBA
Ramiro Quinta- Laboratory of Molecular Neuropharmacology- FMED- UBA
Francisco Urbano – Laboratory of Molecular Neuropharmacology-FCEN- UBA
Lorena Rela -Department of Physiology and Biophysics – FMED-UBA
Gustavo Murer - Department of Physiology and Biophysics –FMED- UBA
Leloir Institute Foundation
Laboratory of regenerative and protective therapies for nervous system
CONICET
Preliminary Results
GAD67
50
*
60
*
40
MAP2
20
10
se
m
0
10
se
m
4s
em
0
4s
em
% celulas GAD67+/Dapi+
GAD67 Dapi
100
control
paciente
80
% celulas MAP2+ / Dapi+
control
paciente
150
MAP2 Dapi
MAP2
GAD67
*p<0,003 Multiple t-test con correccion para comparaciones
multiples del metodo de Holm Sidak
Preliminary Results
GFAP Dapi
GFAP
*
control
paciente
60
40
20
control
pacient
e
150
% celulas DCX+/Dapi+
*
80
*
100
*
50
DCX
ASTROCYTE MARKER
10
se
m
10
se
m
*p<0,02 Multiple t-test con correccion para comparaciones
multiples del metodo de Holm Sidak
4s
em
0
0
4s
em
GFAP
% celulas GFAP+/Dapi+
100
DCX Dapi
DCX
*p<0,00006 Multiple t-test con correccion para comparaciones
multiples del metodo de Holm Sidak