ARVO 2016 Annual Meeting Abstracts
361 Retinal Stem Cells and Transplantation
Tuesday, May 03, 2016 3:45 PM–5:30 PM
6B Paper Session
Program #/Board # Range: 3766–3770
Organizing Section: Retina
Program Number: 3766
Presentation Time: 3:45 PM–4:00 PM
Generation of retinal photoreceptors from cGMP-Manufactured
Human IPSC line
Jie Zhu, Helen Cifuentes, Joseph Reynolds, Deepak A. Lamba.
Ophthalmology, Buck Institute for Research on Aging, Novato, CA.
Purpose: Retinal degeneration often results in the loss of
photoreceptors, which leads to permanent vision loss. Generating
transplantable photoreceptors using human induced pluripotent stem
cells (iPSCs) to replace lost or dysfunctional photoreceptors holds a
promise to treat a variety of retinal degenerative diseases. Developing
effective methods to produce retinal cells including photoreceptors
using available cGMP-manufactured human iPSC lines is a critical
step for advancing cell replacement therapy to clinical application.
This study aims to make transplantable photoreceptors using a
cGMP-manufactured iPSC line. The generated retinal cells were
tested for their differentiation capability and integration in a host
mouse retina.
Methods: An iPSC line derived in cGMP-compliant conditions was
obtained and used to generate retinal cells via a modified version of
our previous directed differentiation protocol (Lamba et al 2006). The
cells were characterized via QPCR and immunocytochemistry (ICC)
at 6 weeks and 3 months after differentiation. GFP labeled retinal
cells were injected into the subretinal space of 4-6 week old recipient
mice. The eyes were collected at 2 months post transplantation for
analysis by ICC.
Results: iPSCs, generated from CD34+ cord blood cells in a cGMP
facility at Lonza Bioscience, were obtained from Dr. Zeng’s lab. They
were induced to optic field stage for 1 week using small molecules to
inhibit Wnt, BMP and TGFβ signaling pathways along with IGF-1 in
culture medium. The cells were then expanded to generate neuroretina and RPE. Neuro-retinal progenitor cells were manually isolated
from RPE and expanded further to a relatively pure population of
retinal neurons including differentiated photoreceptors by 3 months
of culture based on expression of PAX6, OTX2, TUJ-1, CRX and
AIPL1. The cells were then analyzed for their ability to integrate
into the ONL layer of host retina in IL2rg null humanized mice. We
observed robust integration of photoreceptors with typical mature
photoreceptor morphology and presence of Otx2, Recoverin, and
Rhodopsin markers in the integrated cells.
Conclusions: This study provides strong evidence that transplantable
photoreceptors can be generated from a cGMP-manufactured human
iPS cell line which could then be fast-tracked to the clinic.
Commercial Relationships: Jie Zhu; Helen Cifuentes, None;
Joseph Reynolds, None; Deepak A. Lamba, None
Support: CIRM Grant RB4-05785
Program Number: 3767
Presentation Time: 4:00 PM–4:15 PM
A novel biosynthetic RPE-BrM (Retinal Pigment EpitheliumBruch’s Membrane) assembly suitable for retinal transplantation
therapy
Andrew J. Lotery1, 6, Gareth Ward1, 2, Philip Alexander1,
David Johnston3, Anton Page3, Angela J. Cree1, Atul Bhaskar4,
Sumeet Mahajan5, 2, Martin Grossel2, J Arjuna Ratnayaka1. 1Clinical
Neurosciences, University of Southampton, Southampton, United
Kingdom; 2Department of Chemistry, University of Southampton,
Southampton, United Kingdom; 3Biomedical Imaging Unit,
University of Southampton, Southampton, United Kingdom; 4Faculty
of Engineering and the Environment, University of Southampton,
Southampton, United Kingdom; 5Institute for Life Sciences,
University of Southampton, Southampton, United Kingdom;
6
Southampton Eye Unit, University Hospital Southampton NHS
Foundation Trust, Southampton, United Kingdom.
Purpose: Clinical trial data suggests that treatment of neovascular
age-related macular degeneration (AMD) with Vascular Endothelial
Growth Factor (VEGF) inhibitors may accelerate RPE atrophy. It is
therefore imperative that a novel solution be found to repair/replace
the damaged RPE-BrM in disease. We tested the hypothesis whether
a novel biosynthetic RPE-BrM assembly is able to meet the manifold
demands of the outer retina, with potential to rapidly benefit patients
at risk of advancing blindness.
Methods: We used a combination of methyl methacrylate and
poly(ethylene glycol) methacrylate P(MMA:PEGM) in a 60:40
ratio to create a synthetic BrM scaffold. Enhanced electrospinning
techniques were used to create a nanofibrous web-like structure
similar to the inner collagenous layer of native BrM.
Primary RPE cells isolated from murine retinas were seeded on the
synthetic BrM scaffold. These RPE-BrM assemblies were maintained
in culture for several months up to 1 year, during which time their
structure, physiology and functionality were assessed using TEM/
SEM and confocal microscopy as well as biochemical studies
Results: Electrospun scaffolds had comparable porosity, diffusional
properties and mechanical strength equivalent to human BrM. It also
formed an excellent substrate on which primary RPE cells could
readily attach and proliferate. Individual RPE cells readily adhered to
underlying fibres with vinculin and focal adhesion kinases showing
points of contact. Long-term RPE cultures on scaffolds expressed
the cell-specific marker RPE65, formed ZO-1 junctional complexes,
and showed specialisation of apical membranes with microvilli and
expression of Na/K ATPase. RPE monolayers also secreted VEGF
preferentially via the basolateral membrane. RPE cells proved fully
functional and expressed MerTK and aVb5 on apical surfaces to
ingest fed photoreceptor outer segments, which were internalised in
a time dependent manner. We then tested the biocompatibility and
integrity of the BrM scaffold by subretinally transplanting them in
rabbit eyes using a custom-made injector.
Conclusions: We show that a fully-functional RPE-BrM assembly
can be made that is suitable for transplantation. This approach has the
potential to bring rapid long-lasting benefits to patients with retinal
diseases such as AMD or Retinitis Pigmentosa, and could also be
adapted for other regenerative treatments.
Commercial Relationships: Andrew J. Lotery, None;
Gareth Ward, None; Philip Alexander, None; David Johnston,
None; Anton Page; Angela J. Cree, None; Atul Bhaskar,
None; Sumeet Mahajan, None; Martin Grossel, None;
J Arjuna Ratnayaka, None
Support: Brian Mercer Charitable Trust, Gift of Sight
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 3768
Presentation Time: 4:15 PM–4:30 PM
Phase I/II clinical trial of human embryonic stem cell (hESC)derived retinal pigmented epithelium (RPE) transplantation in
Stargardt disease (STGD): One-year results
Manjit S. Mehat1, 2. 1UCL Institute of Ophthalmology, London, United
Kingdom; 2NIHR Moorfields Biomedical Research Centre, London,
United Kingdom.
Purpose: Stargardt disease is the most prevalent juvenileonset inherited maculopathy. Defects in the gene ABCA4 lead
to accumulation of toxic vitamin A derivatives in cells of the
retinal pigment epithelium (RPE), causing RPE dysfunction and
degeneration. Retinal function may be protected or promoted
by provision of a replenished population of RPE cells. Human
embryonic stem cells (hESCs) provide one source of RPE cells for
transplantation. Our aim was to investigate the safety and tolerability
of subretinal transplantation of a suspension of hESC-derived RPE
cells in advanced Stargardt disease.
Methods: We included 12 participants (aged 34 - 45 years) with
clinical and electroretinographic features of advanced Group 3
Stargardt disease. Following pars plana vitrectomy we injected
subretinally a suspension of hESC-derived RPE cells at doses of 50
K, 100 K, 150 K and 200 K cells. Participants were administered
systemic immunosuppression until 3 months after the transplant
procedure. We assessed systemic and ocular safety, indicators of cell
survival and retinal function for 12 months.
Results: We observed the development of areas of subretinal
hyperpigmentation in all participants, suggesting survival
and engraftment of hESC-derived RPE cells. The extent of
hyperpigmentation correlated positively with the dose of cells
administered (R2=0.58, p<0.05). Hyperpigmentation was associated
with areas of both hypo- and hyper-autofluorescence. We identified
no evidence of tumorigenicity, immune adverse events or other
serious safety concerns related to the transplanted cells. Assessment
of ETDRS visual acuity, microperimetry, static full field perimetry,
colour vision testing and electoretinography demonstrated no
significant loss of visual function in the study eye of any participant.
Conclusions: Subretinal transplantation of hESC-derived RPE cells
in Stargardt disease appears safe and well tolerated up to 12 months.
The favourable safety profile supports the prospect of further studies
to investigate the potential for benefit in less advanced disease.
Commercial Relationships: Manjit S. Mehat, None
Support: MRC MR/K024000/1
Clinical Trial: NCT01469832
Program Number: 3769
Presentation Time: 4:30 PM–4:45 PM
Transplantation of Autologous induced Pluripotent Stem CellDerived Retinal Pigment Epithelium Cell Sheets for Exudative
Age Related Macular Degeneration : A Pilot Clinical Study
Yasuo Kurimoto1, 2, Yasuhiko Hirami1, 2, Masashi Fujihara1, 2,
Chikako Morinaga3, 1, Midori Yamamoto1, 2, Kanako Fujita4,
Sunao Sugita3, 1, Michiko Mandai3, 1, Masayo Takahashi3, 1.
1
Ophthalmology, Institute for Biomedical Research and Innovation
Hospital, Kobe, Japan; 2Ophthalmology, Kobe City Medical Ctr Gen
Hosp, Kobe, Japan; 3Laboratory for Retinal Regeneration, RIKEN
Center for Developmental Biology, Kobe, Japan; 4Clinical Support
Unit, Institute for Biomedical Research and Innovation Hospital,
Kobe, Japan.
Purpose: Recently developed induced pluripotent stem cell (iPSC)
s are a promising donor source for regenerative medicine. We report
the first-in-human case of iPSC therapy in which iPSC-derived retinal
pigment epithelium (RPE) cells were transplanted to a patient with
exudative age-related macular degeneration (AMD).
Methods: A patient with advanced exudative AMD in whom the
current standard treatments had not been effective was recruited. A
small piece of skin was collected from the patient’s arm and used
to generate iPSCs. The iPSCs were differentiated into RPE sheets
suitable for transplantation. After the assessments of safety and
quality of the iPSC-derived RPE, the RPE sheet was subretinally
transplanted following surgical removal of the neovascular tissue.
The patient was monitored by systemic and ophthalmic examinations
for a year after the surgery. The primary outcome to be assessed in
this pilot study is the safety of this therapeutic protocol.
Results: An autologous iPSC-derived RPE sheet was successfully
transplanted. The submacularly transplanted RPE sheet survives well
without any findings of immune rejection nor adverse proliferation,
and any significant adverse events associated with the therapeutic
protocol were not observed for a year. Retinal imaging examinations
showed improvement of the pre-existed exudative change. Bestcorrected visual acuity was maintained at 0.09 (=18/200) without
additional anti-VEGF therapy and the score of VFQ (Visual Function
Questionnaire)-25 improved from 40.7 to 58.3.
Conclusions: The first-in human iPSC-based transplantation was
carried out successfully in a patient with exudative AMD. The
primary endpoint, safety of the therapeutic protocol, was attained
after a year.
Commercial Relationships: Yasuo Kurimoto, None;
Yasuhiko Hirami, None; Masashi Fujihara, None;
Chikako Morinaga, None; Midori Yamamoto, None;
Kanako Fujita, None; Sunao Sugita, None; Michiko Mandai,
None; Masayo Takahashi, None
Support: AMED(JAPAN) Grant 15bk0104006h0003
Clinical Trial: http://www.umin.ac.jp/ctr/index-j.htm,
UMIN000011929
Program Number: 3770
Presentation Time: 4:45 PM–5:00 PM
Studies of Structure and Function in Whole Eye Transplantation
Kia M. Washington1, 2, Yang Li3, Chiaki Komatsu1,
Maxine R. Miller1, 4, Yolandi van der Merwe4, 5, Gadi Wollstein4, 5,
Valeria L. Fu4, Vijay S. Gorantla1, Kevin C. Chan4, 5,
Joel S. Schuman4, 5. 1Department of Plastic Surgery, University
of Pittsburgh, Pittsburgh, PA; 2VA Pittsburgh Healthcare System,
Pittsburgh, PA; 3Department of Plastic and Reconstructive Surgery,
Xijing Hospital, Fourth Military Medical University, Xi'an, China;
4
Department of Ophthalmology, University of Pittsburgh, Pittsburgh,
PA; 5Department of Bioengineering, Swanson School of Engineering,
University of Pittsburgh, Pittsburgh, PA.
Purpose: The purpose of this study is to evaluate the structure
and function of our orthotopic whole eye transplant model by
assessing intraocular pressure (IOP), aqueous humor dynamics
using gadolinium (Gd)-enhanced MRI, optic nerve structural
integrity with diffusion tensor MRI (DTI) and functional return via
electroretinography (ERG).
Methods: Syngeneic transplants were performed in 5 Lewis (RT1l)
rats. IOP measurements were made using a TonoLab rebound
tonometer. MRI: 0.3mmol/kg Gd-DTPA (Magnevist) was injected
intraperitoneally after one T1- weighted image at baseline was
acquired. Four animals were scanned at 3 weeks after transplantation,
and 1 animal was scanned at 10 weeks after transplantation.
Scans were performed using a 9.4-Tesla/31-cm Varian/Agilent
scanner. ERG: Rats were placed in a black box overnight. An ERG
system was integrated with a Dell PC equipped with a fast DSP
microprocessor. Recording electrodes gently contacted the corneal
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
surface of eyes precoated with a 2.5% hydroxypropyl-methylcellulose
solution. A subdermal needle electrode served as common reference
while another subdermal needle electrode was inserted at the base of
the left leg. A Ganzfield delivered light stimuli with various stimulus
strengths.
Results: IOPs of the naive and transplanted eye were 15.9±3.1
mmHg and 16.5±3.2 mmHg, respectively. At 3 weeks after
transplantation, the right anterior chamber (AC) had a similar time to
peak but a significantly lower peak intensity and lower initial increase
rate than the left AC. At 10 weeks, the right AC had comparable
peak intensity to the left AC. Limited Gd enhancement was observed
in the vitreous with no significant difference between left and
right eyes (two- tailed paired t-tests, p>0.05). T2-weighted images
showed the donor optic nerve had comparable morphology with
the uninjured intraorbital optic nerve at 3 weeks. DTI quantitation
of the right injured optic nerve showed significantly lower FA and
axial diffusivity by 54±6.1% and 24.9±5.7%, respectively, and a
significant increase radial diffusivity by 83±29.5% compared to
the left uninjured optic nerve (two-tailed paired t-tests, p<0.05).
ERG revealed the lack of electrical responses to light stimuli in the
transplanted eye.
Conclusions: A whole eye transplant model was established revealing
the maintenance of aqueous humor dynamics, IOP and preserved
integrity of the blood-ocular barriers of the transplanted eye.
Commercial Relationships: Kia M. Washington, None;
Yang Li, None; Chiaki Komatsu, None; Maxine R. Miller,
None; Yolandi van der Merwe, None; Gadi Wollstein, None;
Valeria L. Fu, None; Vijay S. Gorantla, None; Kevin C. Chan,
None; Joel S. Schuman
Support: Office of the Assistant Secretary of Defense for Health
Affairs under Award No. W81XWH-14-1-0421
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.