Advances for stem cell research in retinal diseases
Raymond CB Wong, PhD
Unit Head, Cellular Reprogramming UnitCentre for Eye Research AustraliaUniversity of MelbourneGuest Professor, Shenzhen Eye Hospital
Email: [email protected]
Centre for Eye Research Australia
Source: Centre for World University Rankings (CWUR) May 2017
• Department of Surgery (Ophthalmology), University of Melbourne
• Royal Victorian Eye & Ear Hospital
Eye&Ear on the Park (UG)
Royal Victorian Eye & Ear Hospital (Lv 7+8)
Baker Heart and Diabetes Institute (Lv 6)
What is a cell?• An organism’s basic unit of structure & function
• All living things are made of cells
• Cells contain information that is inherited (DNA/genes)
DNA Expressed genes
Protein
What is a cell?• An organism’s basic unit of structure & function
• All living things are made of cells
• Cells contain information that is inherited (DNA/genes)
• Levels of organisation:
– organism, organs, tissues, cells, DNA=> proteins
What is a stem cell?
• Can self-renew: reproduce itself
• Can differentiate: give rise to specialized cells
Handbook of stem cells, Elsevier, R. Lanza, EditorFrom Stem Cell Teacher Kit, 2010(Australian Stem Cell Centre)
Different types of stem cells: Multipotent vs pluripotent
From Stem Cell Research FoundationFrom Sarah Wray
Induced pluripotent stem (iPS) cells
• Can proliferate indefinitely in the lab: unlimited cell source
• Pluripotent: can give rise to any cells type in the body
Cell reprogramming
• The genes in a cell dictate the identity of the cells
• Control genetic signals in a cell
‘reprogram’ cell identity
Cell type A: Cell type B:
reprogramming
Gene A
Gene B
Gene C
Gene D
Gene A
Gene B
Gene C
Gene D
ON
ON
OFF
OFF
ON
ON
ON
ON
reprogramming
Cell reprogramming
• The genes in a cell dictate the identity of the cells
• Control genetic signals in a cell
‘reprogram’ cell identity
Skin cells iPS cells
reprogramming
Oct4
Sox2
Klf4
C-Myc
Oct4
Sox2
Klf4
C-Myc
OFF
OFF
OFF
OFF
ON
ON
ON
ON
reprogramming
Nobel prize 2012:
Patient-specific
stem cells
(iPS cells)
Eye cells
Patient
Cell
therapy
DifferentiationReprogramming
Drug screeningSkin cells
Disease modelling
Potentials of induced pluripotent stem (iPS) cells:
Corrected
eye cells
Genetic
correction
?
Reprogramming to make iPS cells
Adult fibroblasts
Reprogramming iPS cells
Skin biopsy
Hair Keratinocytes
Integration-free episomal
reprogramming method:
OCT4, SOX2, KLF4, L-MYC,
LIN28, shRNA for p53
(Hung, Pebay, Wong 2015 JOVE)
Characterization of keratinocyte-derived iPS cells
Oct4
TRA160
Nanog
Pluripotent markers:
4 5 6 10 11 12
KiPSC clones
EBNA-1
PCR: Karyotype:
SMA GATA4 Nestin
Embryoid
bodies
Mesoderm Endoderm Ectoderm
NR
m epi-
duct
Teratoma
Differentiation assays:
(Piao et al. Wong*, Ko*, 2014 Stem Cell Trans Med, *=equal last author)
iPS cell-derived cardiomyocytes:
Max Lim, O’Brien Institute
Degeneration in retinal diseases:
Adapted from http://www.bem.fi/book/28/28.htm
Leber’s Hereditary Optic
Neuropathy (LHON),
Glaucoma
Age-related macular
degeneration (AMD)
Retinitis pigmentosa
Diseases:Retina structure:
Develop methods to turn stem cells into retinal cells
Adapted from http://www.bem.fi/book/28/28.htm
Retina structure:
Photoreceptors
Retinal ganglion cells (RGC)
Retinal pigmented epithelium (RPE)
Turning human pluripotent stem cells into RPE
Lidgerwood et al., Wong..., Pebay (2016) Stem Cell Rev (IF = 3.6)
3D retinal organoid differentiation• ‘Mini-eye’: self-organising optic cup differentiation
(Nakano et al 2012 cell stem cell)
RG
CR
PE
Ph
oto
rece
pto
r
Using iPS cells to study eye diseases
Adapted from http://www.bem.fi/book/28/28.htm
Glaucoma, Leber’s
Hereditary Optic
Neuropathy (LHON)
Age-related macular
degeneration (AMD)
Retinitis pigmentosa
Diseases:Retina structure:
• Characterized by loss of optic nerve cells
• Most common mitochondrial DNA (mtDNA) disease, affecting ~ 1 in 30000 individuals, predominantly young males
• Central vision loss occurs usually around teenage to early twenties
• All LHON cases are caused by mutation in mtDNA encoding for mitochondria Complex I subunits => Precise mechanism of how RGCs die is not known
• Currently no effective treatment for LHON patients *
• No clinical relevant model to study LHON disease
Designed by Brian Ashton, 20 yrs old
LHON patient
(http://thegenetichouse.wordpress.com)
Case study: Leber’s hereditary optic neuropathy (LHON)
Understanding LHON using iPS cells• Extremely difficult to obtain RGC samples from living patient to study
LHON disease
• Currently no RGC cell lines available
• Potentials of iPS cells”
– ‘Disease-in-a-dish’: understand how RGC loss occurs in LHON, which is crucial to developing new treatment
Patient-specific
iPS cells Optic nerve cells
Study the disease
mechanism
Generation of LHON-iPS cells• Generate iPS cells using fibroblasts from healthy controls and LHON
patients
Control iPSC
mt.4160T>C mt.14484T>C
LHON iPSC
Control iPS
cells
OCT4 TRA160 OCT4 TRA160
LHON iPS
cells
Immunocytochemistry
Genotyping mt.11778 G>A (Rev)
Hung S et al, Wong R (2016) Aging
Developing RGC differentiation method
DAPINEFM
BRN3A
F
Gill K et al. Wong R (2016) Sci Rep
C D
+ TTX
B
Electrophysiology:
Increased cell death in LHON optic nerve cells
Cell death in optic nerve cells:
RG
C a
po
pto
sis
(TU
NE
L p
os
itiv
e c
ell
s %
)
Co
ntr
ol
LH
ON
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
**
• For LHON, the cybrid technology can be used to replace mutated mitochondria with healthy donor mitochondria
N
N
deplete mitochondria
(R6G)
LHON cell
Healthy donor cell Enucleation
(Cytochalasin B)
mm
mmm
N
Fusion N
Cybrid
Genetic correction using mitochondrial replacement
Ian Trounce (CERA)
Correction of LHON mutations rescue optic nerve cell death in LHON
mt.4160 T>C
Corrected cybrids
mt.14484 T>C
Parental LHON fibroblast
Donor keratinocytes
RG
C a
po
pto
sis
(TU
NE
L p
os
itiv
e c
ell
s %
)
Co
ntr
ol
LH
ON
Co
rrecte
d c
yb
r id
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
****
Cell death in optic nerve cells:
Stem cell research on eye diseases• Disease modelling
– Macular degeneration: Galloway et al (2017) PNAS
– Best disease: Singh et al. (2013) Human Molecular Genetics
– Retinitis pigmentosa: Jin et al. (2011) PLoS ONE
Tucker et al. (2013) eLife
– Glaucoma: Minegishi et al. (2013) Human Molecular Genetics
Tucker et al. (2014) J Stem Cell Res Ther
– Bietti’s crystalline dystrophy: Hata et al. (2018) PNAS
Plu
rip
ote
nt
stem
cel
ls
Drug discoveryCurrent challenges:
• Current cost of drug discovery is very expensive and takes a long time
– >10 years in drug discovery and development
– >2 billion USD development cost
– ~5-10% success rate for experimental drugs to make it to the clinic
• iPS cells can potentially fast-track the process of drug discovery and testing with clinical relevant model
Ko 2014 SCTM
Drug screening using iPS cells
Haston et al 2016 ARPT
e.g. Familial Dysautonomia, ALS
Eg: Spinal muscular atrophy
Eg: Cystic fibrosis
Cell therapy• Cell therapy is especially helpful in diseases where affected cells are
‘damage beyond repair’
• Potential of iPS cells in for cell therapy:
– Replace cells damaged by the disease
– Unlimited source of cells
– Autologous transplantation *
= minimal risk of graft rejection
Cell therapy• Cell therapy is especially helpful in diseases where affected cells are
‘damage beyond repair’• Potential of iPS cells in for cell therapy:
– Replace cells damaged by the disease– Unlimited source of cells– Autologous transplantation *= minimal risk of graft rejection
• Clinical trials: Macular degeneration– Embryonic stem cell-derived RPE
• [Ocata/Astellas Institute for Regenerative Medicine]: Schwartz et al. (2012) The Lancet
• [London Project to cure blindness]: da Cruz et al. (2018) Nat Biotech• [Cell Cure Neurosciences]: Phase I/IIa• [USC/Regenerative Patch Technologies]: Phase I/IIa
– iPS cell-derived RPE• [RIKEN- Takahashi Lab]: Kamao et al. (2013) Stem Cell Reports,
Mandai et al. (2017) NEJM
Cell therapy for AMD:
iPSC therapy for wet-type AMD:
From RIKEN
Cell therapy for AMD:
iPSC therapy for wet-type AMD:
Outcome for Phase I/IIa trials:• Transplanted cells integrated and
survived• Generally safe• Some vision improvement in some
patients (RIKEN, ACT, LPCB)
From RIKEN
Stem Cell Teacher Kit, 2010 (Australian Stem Cell Centre)
Patient-specific
stem cells
(iPS cells)
retinal cells
Patient
Cell
therapy
DifferentiationCell reprogramming
Drug screeningSkin cells
Disease modelling
Potentials of cell reprogramming / induced pluripotent stem (iPS) cells:
Corrected
retinal cells
Genetic
correction
?
Patient-specific
stem cells
(iPS cells)
retinal cells
Patient
Cell
therapy
DifferentiationCell reprogramming
Drug screeningSkin cells
Disease modelling
Potentials of cell reprogramming / induced pluripotent stem (iPS) cells:
Corrected
retinal cells
Genetic
correction
?
Direct
reprogramming
Using cell reprogramming for regenerative medicine
Fang et al., Wong (2018) Front Cell Neurosci (5 yr IF= 4.9)
Extracted from De Lazaro et al. 2016 Stem Cell Rev Rep
Glial cells -> retinal neurons: Ueki (2015) PNAS, Jorstad (2017) NatureYao (2018) Nature
Direct reprogramming in vivo:
Using direct reprogramming to promote photoreceptor regeneration
Adapted from http://www.bem.fi/book/28/28.htm
Leber’s Hereditary Optic
Neuropathy (LHON),
Glaucoma
Age-related macular
degeneration (AMD)
Retinitis pigmentosa
Diseases:Retina structure:
Photoreceptors
• 1000 pixel => sufficient for blind patients to recognise faces
Using direct reprogramming to promote photoreceptor regeneration
Adapted from http://www.bem.fi/book/28/28.htm
Leber’s Hereditary Optic
Neuropathy (LHON),
Glaucoma
Age-related macular
degeneration (AMD)
Retinitis pigmentosa
Diseases:Retina structure:
Photoreceptors
• 1000 pixel => sufficient for blind patients to recognise faces• ~5% reprogramming efficiency would generate 1500
photoreceptors in the macula (1500 pixels)
Using direct reprogramming to develop therapy to regenerate photoreceptors
reprogramming
photoreceptorsMuller cells
In the lab dish
In animal models
Reprogrammingfactors
Rat retina
reprogramming
Reprogrammingfactors
Muller cells photoreceptors
Assess improvement of retinal functions in animal model of
retinitis pigmentosa
Patient-specific
stem cells
(iPS cells)
retinal cells
Patient
Cell
therapy
DifferentiationCell reprogramming
Drug screeningSkin cells
Disease modelling
Summary of stem cells in eye research:
Corrected
retinal cells
Genetic
correction
?
Direct
reprogramming
External collaborators (National)Mark Gillies (Save Sight/Uni Sydney)David Mackey (Lions Eye Institute)Joseph Powell (Gavan/Uni Queensland)Max Lim (O’Brien)Karina Needham (Uni Melb)Sam Lukowski, Quan Nguyen (Uni Queesnland)Jafar Jabbari (AGRF)Trevor Lamb (ANU)Rick Liu (Uni Tasmania), Camden Lo (Lo LLC)
External collaborators (International)George Wang (Chinese Academy of Science)Alexei Sharov (NIH, USA)Jane Sowden (UCL, UK)Peter Donovan (UC Irvine, USA)Minoru Ko (Keio Uni, Japan)Martin Pera (Jacksons Lab, USA)
Cellular Reprogramming UnitCrystal Nguyen, Layal El Wazan, Daniel Urrutia, Sheridan KeenePast members: Lucy Fang, Christine Tan
Acknowledgements:
Centre for Eye Research Australia: Alex Hewitt, Sandy Hung (CERA) Chi Luu, Robyn Guymar (CERA)Lions Eye Bank (CERA)Alice Pebay (UniMelb), Ian Trounce (CERA)
CRU & CGU @ Baker node
Kel and Rosie Day Foundation
Support:
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