Advances for stem cell research in retinal diseases Research Australia.pdf · Cell therapy • Cell therapy is especially helpful in diseases where affected cells are ‘damage beyond

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:

http://www.bem.fi/book/28/28.htm

Develop methods to turn stem cells into retinal cells


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


Glaucoma, Leber’s

Hereditary Optic

Neuropathy (LHON)

Age-related macular

degeneration (AMD)




• 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)

http://thegenetichouse.wordpress.com/

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


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



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



Neuropathy (LHON),

Glaucoma

Age-related macular

degeneration (AMD)



Photoreceptors

• 1000 pixel => sufficient for blind patients to recognise faces


Using direct reprogramming to promote photoreceptor regeneration



Neuropathy (LHON),

Glaucoma

Age-related macular

degeneration (AMD)



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



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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Advances for stem cell research in retinal diseases Research Australia.pdf · Cell therapy • Cell therapy is especially helpful in diseases where affected cells are ‘damage beyond