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The University of Hong KongLi Ka Shing Faculty of Medicine香港大學李嘉誠醫學院
Press ConferenceApril 18, 2018
HKU Discovers the Application of Human Induced Pluripotent Stem Cells in Precision
Medicine for Hereditary Diseases
Professor David Siu Chung-wahClinical Professor of Department of Medicine
Speaker
“An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.”*
– Enables physicians to tailor medical treatment for each patient
– Supports the development of molecularly targeted drugs based on biologic pathways
– Identifies at-risk populations for targeted prevention prior to disease onset
*NIH - https://ghr.nlm.nih.gov/primer/precisionmedicine/definition
What is Precision Medicine?
In precision medicine, the patients' disease risks, prognoses, and treatment responses can be predicted based on the behaviors of their hiPSC derivatives in cell culture
Patient-specific hiPSC derivatives recapitulate the phenotypes of their in vivo counterparts
Differences in patients' clinical phenotypes are the result of their unique DNA sequences
Differential gene expression patterns cause different cellular and tissue phenotypes
Role of Human Induced Pluripotent Stem Cell (hiPSC) Technology in Precision Medicine
Human Induced Pluripotent Stem Cell (hiPSC)-Based Platforms for Drug Development
28
Skin biopsy and dermal fibroblast
expansion
bFGF supplement
2-10 7 14 21
Emergence of ES cell-like colonies
Manual picking and subculturing of iPS cell
colonies
ROCK kinase inhibitorLentiviral Transduction
0
Switch to MatrigelTM coated
plate
1
Day
Reprogramming genes ROCK kinase inhibitor
Skin cells
Micro-dissection & clonalexpansion
hiPSCs
Lai WH, ... Tse HF, Siu CW: Cell Reprogram. 2010 Dec;12(6):641-53.
Animal Product-Free hiPSC Production
Embryoid bodies Smooth muscle cells Skin and nerve cells
Liver cells Heart cells Blood vessel cells
Lai WH, ... Tse HF, Siu CW: Cell Reprogram. 2010 Dec;12(6):641-53.
Branching into various cell types of the body
Beating Heart Muscle Generated from hiPSC
• Alternative splicing of LMNA gene generates lamin A and C,the intermediate filament protein of nuclear lamina.
• Serve as a matrix to maintain chromosome and genomeintegrity
• Mutations in LMNA referred to as “laminopathies, whichcause Hutchinson Gilford Progeria (premature agingsyndrome) and muscular dystrophy, to familial dilatedcardiomyopathy (DCM).
Laminopathies
Hutchinson-Gilford Progeria Syndrome In HGPS patients the cell nucleus has dramatically aberrant morphology (Scaffidi et al., 2005).
Laminopathies are rare human degenerative disorders with a wide spectrum of clinical phenotypes.
Laminopathies
0-20 years
Atrial fibrillation 30-60 years
Cardiomyopathy VT/VF 40-60 years
2nd to 3rd AV nodal block 20-40 years
Asymptomatic Conduction block 1st degree AV block•Sick Sinus Rhythm
SIU CW Aging (2011)
LAMIN A/C Related Cardiomyopathy
Pedigree of LMNAR225X/WT probands with two females (II-5 & II-9) and one male (II-7)
WT:GCACAGAACACCTGGGGC
MT:GCACAGAACACTGGGGC
heterozygous
frameshift mutation
T518fs
Deletion of ‘c’
Frameshift
WT: ATGCAGCAGCAG
MT: ATGCAGTAGCAG
heterozygous
nonsense mutation
Q354X
C to T
Q
stop
TAG premature stop
WT: CGTCATGAGACCCGACTGGTGG
MT: CGTCATGAGACCTGACTGGTGG
heterozygous
nonsense mutation
R225X
C to T
stop
TGA premature stop
Schematic diagram of LMNA/C structure presenting mutation sites of our enrolled patients
Head Coil 1A Coil 1B Coil 2 Tail
1 240 244 350
35 70 82
644
1 240 244 350
35 70 82
572
Lamin A
Lamin C
1 225
35 70 82
R225X
1 240 244 354
35 70 82
Q354X
1 240 244 350
35 70 82
T518fs
2 3 4 5 6 7 8 9 10 11 121
c.675C>T c.1062C>T c.1554delC
LMNA
Lamin A HeadHead
Coil1A Coil 1B Coil 2 Tail
NLS
Tru
nca
ted
Lam
inA
518
Schematic Diagram Illustrating the LMNAMutations Involved in Current Study: Nonsense
and Frame-Shift Mutations in LMNA
Table 1. Cardiac manifestations in affected subjects bearing LMNA mutation in present studyAbbreviations: 1 HB: first degree heart block; 2 HB: second degree heart block; 3 HB: third degree heart block; AF: Atrialfibrillation; AICD: automatic implantable cardioverter defibrillator; PPB: permanent pacemaker; AV block: atrio-ventricular block;CHB: complete heart block; PR: P-R interval; VT: ventricular tachyarrhythmia, DCM: dilated cardiomyopathy.* Three probands were come from the same family.
Cardiac Manifestations (age of diagnosis in years)
Affectedsubjects
Sex
heartblock AF VT/VF Cardiomyopat
hyAICD/Pacemaker Age of death
SF5* (II-7)LMNAR225X/WT M CHB (49) + (49) + (50) - PPM (49);AICD
(52) (57)
SF29*(II-9)LMNAR225X/WT F CHB (48) + (48) - - PPM (49);AICD
(53) -
SF36*(II-5)LMNAR225X/WT F CHB (51) + (52) + (60) + DCM (60) ICD (60) -
SF11LMNAframeshift/
WTM 3 HB (46) + (49) - - Pacemaker (50) -
SF26LMNAE422X/WT M CHB (50) + (54) + (54) + DCM (57) ICD (?) (64)
SF30LMNAQ534X/WT M CHB (50) + (50) + (56) + DCM (50) PPM: AICD (50) (56)
SF39LMNAT918fs/WT M CHB (43) + (43) + (47)) + DCM (47) AICD (47) -
ActininLMNA
LMNAwt/wt
ActininLMNA
LMNAR225X/wt
ActininLMNA
LMNAwt/wt +GFP
LMNAwt/wt + shLMNA
ActininLMNA
ActininLMNA
ActininLMNA
ActininLMNA
LMNAwt/wt
LMNAR225X/wt
ActininLMNA
ActininLMNA
LMNAwt/wt +GFP
LMNAwt/wt + shLMNA
基線 電刺激
hiPSC Heart muscle cells from patients with Cardiac Laminopathy exhibit nuclear blebbing
upon electrical stimulation
SIU CW Aging (2011)
PTC 124 Chemical Structure
• PTC124 (Ataluren) is a novel small molecular CFTR-G542X nonsense allele inhibitor.• In safety pharmacology studies in rats and dogs, oral administration of PTC124 (Ataluren) induces
no adverse neurological, pulmonary, or cardiovascular effects at doses through 1500 mg/kg.• In toxicology studies in rats and dogs at oral doses through 1500 mg/kg for 28 days, PTC124
(Ataluren) has shown good tolerability.• FDA approved a new indication for orphan drug PTC 124 (made by PTC Therapeutics, Inc.),
allowing its use in the treatment of Duchenne muscular dystrophy (MD) caused by a nonsensemutation in the dystrophin gene.
(Nature, Vol 447 2007)
PTC124 (Ataluren)
X
50S
30S
F-MET
ARGGLY SER PRO THR
50S
30S
F-MET
ARGGLY SER PRO
A U G C G C G G A U C C C C C A C C U G A
5’ 3’Codon 1(f-MET/Start)
Codon 2(ARG)
Codon 3(GLY)
Codon 4(SER)
Codon 5(PRO)
Codon 6(THR)
Codon 7(STOP)
X X X X X X X X X
THR
A U G C G C G G A U C C C C C A C C U G A
5’ 3’Codon 1(f-MET/Start)
Codon 2(ARG)
Codon 3(GLY)
Codon 4(SER)
Codon 5(PRO)
Codon 6(THR)
Codon 7(STOP)
X X X X X X X X X
PTC124
X X XX
PTC124 reverses or alleviates nonsense mutation
Lamin A/C
ACTB
Lamin A/C
ACTB
Lamin A/C
ACTB
Lamin A/C
ACTB
Lamin A/C
ACTB
Lamin A/C
ACTB
Lamin A/C
ACTB
Skin Fibroblast iPSC-CMC
PTC (µM)0 50
PTC (µM)0 50
LMNAR225X/wt
LMNAT518fs/wt
LMNAwt/wt
LMNAQ354X/wt
Lamin A/C
ACTB
Effects of PTC124 on Expression of Lamin A/C Proteins in Dermal Fibroblasts and hiPSC-
Derived Cardiomyocytes
LMNAWT/WT
CT
RE
lect
ric
ally
st
ress
edE
lect
ric
ally
str
ess
ed+
PT
C12
4
100 µm
100 µm
100 µm
LMNAR225X/WT
100 µm
100 µm
100 µm
LMNAQ354X/WT
100 µm
100 µm
100 µm
LMNAT518fs/WT
100 µm
100 µm
100 µm
Nuclear blebbing in the hiPSC-derived cardiomyocytes
DNA content (PI)
E-StimControl
Ap
op
toti
c c
ell
(TU
NE
L p
osi
tive
: B
rdU
-FIT
C)
FITC
Log
LMN
AR22
5X/w
t LM
NAT5
18f
s/w
t
LMN
Awt
/wt
E-Stim+ PTC
LMN
AQ35
4X/w
t
6.32 ± 1.72
33.78 ± 1.72
28.20 ± 13.70
59.85 ± 13.79
7.48 ± 3.94
69.96 ± 7.49
17.38 ± 7.23
43.78 ± 15.30
8.78 ± 3.38
20.6 ± 3.91
26.13 ± 5.75
54.58 ± 16.28
Evaluation of TUNEL-Positive Apoptotic Cell in Electrically Stressed and PTC124-Treated
Cardiomyocytes
Co
ntr
actil
ity(%
)
D
S
DL
Ds: Duration of shorteningDL: Duration of Lengthening
Cell shortening: Video based edge detection used to record cell shortening
Peak High
Baseline
Field electrical pacing at 40 Vat the frequency of 0.5 Hz, 1 Hz, 1.5 Hz and 2 Hz
LMNAWT/WT
PTC124CTR
0.0 0.5 1.0 1.5 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+i(n
M)
40
45
50
55
60
65
Cel
l le
ng
th (
uM
)
0.0 0.5 1.0 1.5 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+i(n
M)
40
45
50
55
60
65
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca
2+
i(nM
)
40
45
50
55
60
65
Ce
ll le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+i(n
M)
40
45
50
55
60
65
Cel
l len
gth
(u
M)
LMNAR225X/WT
PTC124CTR
0.0 0.5 1.0 1.5 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
20
30
40
50
60
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
20
30
40
50
60
Cel
l len
gth
(u
M)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
20
30
40
50
60
Ce
ll le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
20
30
40
50
60
Ce
ll le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+i(n
M)
20
25
30
35
40
45
50
55
60
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+i(n
M)
20
25
30
35
40
45
50
55
60
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca
2+ i(n
M)
20
25
30
35
40
45
50
55
60
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca
2+ i(n
M)
40
41
42
43
44
45
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
20
25
30
35
40
45
50
55
60
Cel
l le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca
2+
i(nM
)
20
25
30
35
40
45
50
55
60
Ce
ll le
ng
th (
uM
)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
Time (s)
Ca2
+i(n
M)
40
45
50
55
60
65
70
Cel
l len
gth
(u
M)
0.0 0.5 1.0 1.5 2.0
20
40
60
80
100
120
140
160
180
200
Time (s)
Ca2
+
i(nM
)
40
45
50
55
60
Cel
l len
gth
(u
M)
0.5
Hz
1 H
z1.
5 H
z2
HzDiastolic Ca2+
concentration
Ca2+ Transient: the calcium indicator,Fura-2 AM was loaded into cells
Am
pli
tud
e
Simultaneous Recording of Cardiac Cell Contractile Force and Calcium Transients of
Single Cardiomyocytes
• Precision medicine is a new trend in modern medicine. It aimsto allow tailing disease treatment and prevention according toindividual variability in genes, environment, and lifestyle foreach person.
• hiPSC technology provides an unique platform for cliniciansand scientists to study the underlying mechanisms of variousdiseases.
• More importantly, while the clinical manifestations are verysimilar in patients with the same disease, hiPSC technologyallow better prediction to clinical responses prior to realclinical trials. This is particularly important for rare diseases,whose number of patients often too small for meaningfulclinical trials.
Conclusions
• In fact, since 2008, we have generated more than 20 disease-specific hiPSC line. This hiPSC bank is a unique platform forinnovative biomedical research and drug development inHong Kong and Mainland China.
• The present work demonstrate the feasibility of hiPSCtechnology in precision medicine for rare disease,representing a step forwards to its clinical applications.
Conclusions