Wednesday, April 30, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 4276-4290 / A424-A438
440. AMD Surgical Therapy Organizing Section: RE Contributing Section: LE
4276 - A424
A Comparison of the Murine RPET Cell Line With Human ARPE19 Cells for
Use in Retinal Transplant Studies
4277 - A425
Gene Expression Patterns of Retinal Progenitor Cells Before and After
Transplantation
T.M. Holmes1, K. Kennelley1, H. Tomita2, D.J. Keegan1. 1Ophthalmology, Mater
Misericordiae Hospital, Dublin, Ireland; 2Biofunctional Science, Tohoku Univ
Biomedical Eng Research Org, Sendai, Japan.
L.A. Kim1, Z. Chen1, B. Thomas1, D. Mock 2, S.R. Sadda1. 1Ophthalmology, Doheny Eye
Institute, Los Angeles, CA; 2USC-CHLA Genome Core, Los Angeles, CA.
Purpose: Age related macular degeneration is a major cause of visual morbidity
world-wide. Retinal cell transplantation is one possible therapy and in recent years
transplants of RPE have been shown to delay photoreceptor degeneration in the RCS
Rat. Basic research in this field has been limited by a failure to accurately identify the
temporal health and survival of grafted tissue. We have previously determined that
only 2-5% of grafted human cells and fewer rat cells survive 5 months post graft. Graft
cell survival is crucial to the success of this research before we can answer questions
on immune responses and long term function.
This project aims to characterise an extended life murine RPE cell line for use in
allogeneic subretinal transplant studies. The mouse is a more versatile animal model
for use in immunological experiments and allows us to take advantage of transgenic
models for later research.
Methods: The RPET cell line was obtained from Dr Hiroshi Tomita in TUBERO, Sendai,
Japan, they were grown in culture at 33 C on collagen coated flasks and their basic
morphology compared to that of the more commonly used human ARPE19 cell line
(ATCC) The RPET cell line has been derived from transgenic C57BL/6 mice harbouring
temperature sensitive simian virus 40 T-antigen. Immunocytochemistry was used to
assess junctional marker ZO-1 (BD Biosciences), adherens junction protein ß-Catenin
(BD Biosciences), and cytokeratins 5&8 and 18 (both from Eurodiagnostica).
Results: Cell morphology is very similar between the RPET and the ARPE19 cell lines
with both showing characteristic RPE expression patterns for ZO-1 and ß-catenin.
Cells grew as contact inhibited monolayers with the RPET cells exhibiting a slightly
more elongated morphology than the ARPE19. Both cell lines expressed cytokeratins
characteristic of epithelial phenotypes (5&8 for all epithelia and 18 for non stratified
epithelia).
Conclusions: Before using a novel cell line for these experiments it is imperative to
assess the characteristics of the cells and how they compare to normal retinal pigment
epithelial cells. By confirming that these cells conform to well established markers
of RPE cell morphology and function we have shown that they are suitable for use
in retinal transplantation studies. These cells will allow future cell survival studies
assessing basic immunological responses to allogeneic subretinal transplantation of
RPE cells in a murine model.
CR: T.M. Holmes, None; K. Kennelley, None; H. Tomita, None; D.J. Keegan,
None.
Support: Mater Misericordiae foundation Grant
4278 - A426
Retinal Pigment Epithelial Cell Culture on Silk Substrate for Retinal Tissue
Transplantation
Purpose: To analyze the gene expression patterns of retinal progenitor cells prior to
and after transplantation in S334ter-line-3 (line-3) rats, a transgenic model of retinal
photoreceptor degeneration.
Methods: Retinal progenitor cells (RPC) were collected from embryonic day 19 (E19)
hPAP/GFP rats. These RPCs were then harvested after cell culture passage 5. They
were subsequently transplanted within the sub-retinal space of 1 month old line-3
rats. Using the Zeiss laser microdissection system, the transplanted layer of RPCs
were gathered. Total RNA was purified (RNeasy Micro Kit, Qiagen, Valencia, Ca) from
RPCs prior to and after transplantation. Gene expression patterns were compared
between pre- and post-transplant RPCs with a rat genome microarray (Rat Genome
230 2.0 Array, Affymetrix Inc., Santa Clara, Ca). Data analysis was performed using the
S-score algorithm to determine statistical significance. The Gene Ontology Database
was used to determine the biological significance of a selection of differentially
expressed genes.
Results: Setting a p-value < 10 -2, genes were selected that were differentially expressed
within pre and post-transplant RPCs. The following biological processes were enriched
within the differentially expressed group of genes: (1) detection of light stimulus (9.23
fold); (2) regulation of exocytosis (7.48 fold); (3) gamma-aminobutyric acid signaling
pathway (6.80 fold); (4) generation of a signal involved in cell-cell signaling (3.40
fold). As an example, genes involved in detection of light stimulus were found to be
differentially expressed including: GNAT2, GRM6, SAG, RGR, GNGT2. Similarly,
we discovered several genes that were differentially expressed within the processes
mentioned above.
Conclusions: Differences in gene expression between retinal progenitor cells before
and after transplantation reveal specific biological processes that differ once RPCs are
transplanted within the subretinal space. These differences highlight the importance
of the subretinal milieu in regulating gene expression within RPCs.
CR: L.A. Kim, None; Z. Chen, None; B. Thomas, None; D. Mock, None; S.R. Sadda,
None.
Support: Foundation Fighting Blindness; Private Funds, Foundation for Retinal
Research, Fletcher Jones Foundation, Walsh Stem Cell Foundation, NIH EY03040
4279 - A427
Over-Expression of Developmentally Regulated Neurotrophins in the Adult
Mouse Retina to Facilitate Integration of Transplanted Photoreceptor Precursor
Cells
S. Cheng, A. Kwan, Z. Barnard, Z. Zainuddin, D. Harkin, T. Chirila. Ophthalmology,
Queensland Eye Institute, Brisbane, Australia.
Purpose: A potential treatment for age-related macular degeneration is retinal pigment
epithelial cell (RPE) transplantation. In this study we evaluated a novel substratum for
RPE culture derived from domesticated silkworm Bombyx mori silk fibroin (BMSF),
both alone or coated with a selection of extracellular matrix (ECM) proteins, and
with or without serum.
Methods: The ARPE-19 cell line was seeded onto tissue culture plastic (TCP), BMSF
membrane alone or BMSF membrane coated with laminin, vitronectin, fibronectin,
a laminin-vitronectin-fibronectin combination (LVF) or collagen-IV. Samples were
cultured under Foetal Bovine Serum (FBS) containing media for 72 hours, then fixed
and nuclei stained with Hoechst and viable cells attached were counted. Experiments
were repeated with serum starved ARPE-19 cells, which were seeded on to the different
substrates, cultured under serum free conditions for 24 hours. Samples were fixed;
nuclei stained and attached cells counted.
Results: ARPE-19 cell growth on BMSF membrane demonstrated no statistical
difference (P>0.05) to TCP under FBS containing culture condition. However, the
ARPE-19 cell count on unmodified BMSF membrane was 60% of that on TCP under
serum free condition (P<0.05). Cell count on ECM protein coated BMSF surpassed
BMSF alone (vitronectin>collagen IV>fibronectin>LVF>laminin), except for laminin
coated. Cell attachment on vitronectin or collagen IV coated BMSF was comparable
to that seen in medium containing FBS on TCP.
Conclusions: ARPE-19 cells can be grown on BMSF membranes. Coating BMSF with
ECM proteins increases attachment under serum free culture conditions. BMSF appears
to be a potential substrate for RPE transplantation and merits further investigation.
CR: S. Cheng, None; A. Kwan, None; Z. Barnard, None; Z. Zainuddin, None; D.
Harkin, None; T. Chirila, None.
Support: Prevent Blindness Foundation
E.L. West1, R.A. Pearson1, Y. Duran1, U.F.O. Luhmann1, S.E. Barker1, A.J. Smith1, J.C.
Sowden2, R.E. MacLaren 3,4, R.R. Ali1,4. 1Division of Molecular Therapy, Institute of
Ophthalmology, UCL, London, United Kingdom; 2Developmental Biology Unit,
Institute of Child Health, UCL, London, United Kingdom; 3Division of Molecular
Therapy, UCL Institute of Ophthalmology and Moorfields Eye Hospital, London,
United Kingdom; 4NIHR Faculty, UCL/MEH London, United Kingdom.
Purpose: Photoreceptor cell transplantation provides a novel therapeutic strategy to repair
the degenerate retina, although greater numbers are required than has been achieved thus
far. Growth factors, such as CNTF, bFGF and IGF-1, provide important, tightly regulated
signals that direct the proliferation and differentiation of progenitors in the CNS, including
the retina, and play a role in regulating neural precursor cell migration. Neurotrophins
may also influence the integration of transplanted photoreceptor precursors. It is therefore
important to see whether modulating neurotrophin levels leads to a greater yield of
integrated photoreceptors.
Methods: CNTF, bFGF or IGF-1 were delivered to the host retina by adeno-associated
virus type 2 (AAV2/2) viral vectors 4 weeks prior to cell transplantation; an AAV2/2 vector
encoding a red fluorescent protein reporter construct was used as a control. Cells from
dissociated P3 neural retinas were transplanted subretinally. At 3 weeks post-injection,
the number of integrated, differentiated photoreceptor cells present in growth factor
treated eyes, was compared to the control treated contralateral eye.
Results: Increased levels of secreted CNTF protein in the host retina led to a significant
decrease in the number of integrated photoreceptor cells. Similarly, increased bFGF
expression had adverse effects, both on the number of integrated photoreceptors and the
survival of unintegrated cells in the subretinal space. Conversely, more integrated cells
were observed in the IGF-1 treated eyes, compared to contralateral controls. Examination
of control eyes demonstrated that AAV2/2 vector administration had no detrimental
effects on precursor cell integration.
Conclusions: Here, we show that photoreceptor precursor cell integration can be
modulated by ectopic expression of growth factors in the adult host retinal environment.
Increased levels of the neurotrophic factors, CNTF, bFGF and IGF-1, had differential
effects on the transplantation and integration of photoreceptor precursor cells in the
adult mouse. CNTF appears to play an inhibitory role in cell integration, possibly due
to respecification of the donor cell population.
CR: E.L. West, None; R.A. Pearson, None; Y. Duran, None; U.F.O. Luhmann, None; S.E.
Barker, None; A.J. Smith, None; J.C. Sowden, None; R.E. MacLaren, None; R.R. Ali,
None.
Support: Medical Research Council UK, Fighting Blindess, Ireland, Royal Society UK,
The Health Foundation UK
Copyright 2008 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4276-4279
Wednesday, April 30, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 4276-4290 / A424-A438
440. AMD Surgical Therapy Organizing Section: RE Contributing Section: LE
4280 - A428
GMP-Compliant Human ES-Derived RPE Cells Rescue Vision in Mouse Models
of Macular Degeneration
S. Wang1, B. Lu1, S. Girman1, C. Malcuit2, L. Vilner2, L. Lemieux2, I. Klimanskaya2,
K. Zhang3, P. Francis1, R. Lund1,3. 1Ophthalmology, Casey Eye Institute, Oregon
Health & Science Univ, Portland, OR; 2Advanced Cell Technology, Worcester, MA;
3
Ophthalmology, Moran Eye Center, Salt Lake City, UT.
Purpose: Stargardt’s macular dystrophy is one of the most frequent forms of juvenile
macular degeneration. It is characterized by progressive accumulation of lipofuscin
in the retinal pigment epithelium (RPE), which leads to atrophy of RPE cells and
followed by photoreceptor death. There is no treatment available yet. Age-related
macular degeneratio is the leading cause of blindness in older individuals and shares
some pathogenic similarities. The presence of animal models, particularly rodents
with diseases homologous or analogous to human disorders, allows investigators to
explore therapeutic approaches that might eventually be applied for human diseases.
Cell-based therapy for eye disease has been shown very effective in limiting the
progress of retinal degeneration and in rescuing vision. Here we examine Good
Manufacturing Practice (GMP)-compliant human ES-derived pigmental epithelium
(hES-RPE) cells in rescue vision in mouse modesl of macular degeneration.
Methods: Both ElovL4+/- (TG3) (University of Utah) and Ccl2-/- (Jackson laboratory) mice
received subretinal injections of human embryonic stem cell-derived RPE cells (50,000/
eye) manufactured under GMP-compliant condition; medium alone and untreated
eyes were used as control. All animals were maintained under oral cyclosporine
A administered in the drinking water. The animal’s acuity was tested 2 weeks, 4
weeks, 6 weeks and 8 weeks after surgery by optomotor responses. Morphologically,
we examined the donor cell distribution by human-specific antibodies, and the
relationships between donor cells and cells of the host retina, as well as looked for
untoward pathological events.
Results: At all time points studied, cell-grafted animals performed consistently
better than sham and unoperated animals by the optomotor test. Histological study
demonstrated that there is no sign of tumor formation or untoward pathological
events.
Conclusions: This is the first study that indicates cell-based therapy can rescue
visual function in mouse models of macular degeneration. This adds to the value
of human embryonic stem cell-derived RPE cells as a potential therapy for macular
degeneration.
CR: S. Wang, None; B. Lu, None; S. Girman, None; C. Malcuit, ACT, E; L. Vilner,
ACT, E; L. Lemieux, ACT, E; I. Klimanskaya, ACT, E; K. Zhang, None; P. Francis,
ACT, C; R. Lund, ACT, C.
Support: Advanced Cell Technology, Foundation Fighting Blindness and the Lincy
Foundation
4281 - A429
Autologous Transplantation of Retinal Pigment Epithelium and PartialThickness Choroid After Mechanical Debridement of Bruch’s Membrane in
Rabbit
Y. Hu, T. Zhang, J. Wu, Y. Li, X. Lu, F. Qian, Z. Ma. Peking University Eye Center,
Peking University Third Hospital, Beijing, China.
Purpose: Autologous transplantation of retinal pigment epithelium (RPE) sheet may
be helpful for the surgical treatment of age-related macular degeneration (AMD). We
developed a new technique for autologous RPE transplantation. The graft is a sheet
of partial-thickness RPE-choroid.
Methods: 27 pigmented rabbits were used for this study. After mechanical debridement
of Bruch’s membrane, sheets of partial thickness RPE-choroid were transplanted to
the subretinal space in 25 rabbits. The animals were examined by fundus photographs
and fluorescein angiographs, and were sacrificed postoperatively at 1, 2, 4, 12, and
24 weeks. Eye cups containing the graft were examined by light microscopy and
immunohistochemistry. In addition, two partial-thickness RPE-choroid sheets were
analyzed by transmission electron microscopy (TEM).
Results: TEM showed partial-thickness RPE-choroid graft consisted of RPE cells,
Bruch’s membrane, choriocapillaries and ruptured middle vessels. Its thickness is
about 50-60µm. Fluorescein angiography revealed neither fluorescein leakage nor
stain at the graft at either early or late phase. Light microscopy revealed that in 17
experiments where the graft survived and the neural retina remained intact; but in 8
experiments with unsuccessful grafts, the neural retina degenerated. The implanted
graft had revascularization and RPE cells were monolayered. In sections that the neural
retina survived where the graft implanted, anti-CRALBP antibody labelled all RPE
cells and rhodopsin in photoreceptor outer segments was positive. Figure showed
that the graft survived at postoperative 2 weeks (A) and 24 weeks (B).
Conclusions: Our study documented the feasibility and the histological outcome
of autologous transplantation of partial-thickness RPE-choroid sheet. In humans,
autologous transplantation of partial-thickness RPE-choroid sheets might become
a treatment for AMD.
CR: Y. Hu, None; T. Zhang, None; J. Wu, None; Y. Li, None; X. Lu, None; F. Qian,
None; Z. Ma, None.
Support: Peking University Third Hospital Clinical and Basic Research Grant
4282 - A430
Transplantation of Pigmented RPE-Like Cells Derived From Human Embryonic
Stem Cells Provides Functional and Structural Rescue in Dystrophic RCS Rats
4283 - A431
Long Term Visual Rescue of GMP-Compliant Human ES-Derived RPE Cells
Transplanted into RCS Rats
R. Alper1A, A. Obolensky1A, M. Idelson1B, I. Hemo1A, R. Yaul1A, B. Reubinoff 1B, E. Banin1A.
A
Ophthalmology, BCenter for Human Embryonic Stem Cells, 1Hadassah-Hebrew
Univ Med Center, Jerusalem, Israel.
B. Lu1, S. Wang1, S. Girman1, P. Francis1, C. Malcuit2, L. Vilner2, L. Lemieux1, R. Lanza2,
R. Lund1,3. 1Casey Eye Institute, Oregon Health Sciences University, Portland, OR;
2
Advanced Cell Technology, 381 Plantation Street, Worcester, MA; 3University Of
Utah, Salt Lake City, UT.
Purpose: Replacement and support of dysfunctional Retinal Pigment Epithelium
(RPE) may be beneficial in AMD and in subtypes of retinitis pigmentosa. Human
Embryonic Stem Cells (hESCs) may serve as an unlimited source for such cells. We
examined survival, function and therapeutic potential of pigmented RPE-like cells
derived from hESCs in the RCS rat model of retinal degeneration.
Methods: Differentiation of hESCs engineered to express eGFP was induced by
culturing embryonic bodies (EBs) in suspension for a minimum of 4 weeks. Clusters of
pigmented cells within the EBs were mechanically dissected and further cultured. These
pigmented cells expressed RPE-specific markers in-vitro and formed “cobble stone”like arrays upon plating on laminin. A suspension enriched with pigmented cells was
injected into the subretinal space of one rat eye. Fellow control eyes were either nontreated or medium-injected. Retinal function was assessed using electroretinography
(ERG) 4 weeks after transplantation. Survival and location of the grafts, expression
of RPE-specific markers, and thickness of the host photoreceptor layer (ONL)were
examined using histological and immunohistochemical techniques.
Results: Dark-adapted (DA) ERG mixed cone-rod responses were significantly
better preserved in eyes that received transplants of hESC-derived RPE-like cells.
At the highest intensity, mean DA b-wave amplitude in RPE-transplanted eyes was
283.3±37.5μV(n=13) versus 158.5±18.1μV in fellow non-treated control eyes (n=13,
p<0.01) and 89.9±14.4μV in medium-injected eyes (n=5, p< 0.01). Large numbers of
transplanted pigmented cells were found under the host RPE, in the sub-retinal space
and occasionally within the retina and vitreous. These GFP+ cells expressed RPEspecific markers including RPE-65 and Bestrophin as well as the tight junction marker
ZO-1. A small precentage expressed the proliferation marker Ki-67. Morphometric
analysis of the host retina in vicinity to grafts showed significant preservation of
the ONL: thickness of 25.9±3.7µm versus 12.4±2.1µm in areas distant from grafts
(p<0.001, n=6).
Conclusions: RPE-like cells derived from hESCs can survive for at least 4 weeks
after transplantation into RCS rat eyes, and maintain expression of RPE-specific
markers. Transplanted eyes show functional and structural rescue. The results support
the potential use of hESC-derived RPE for the treatment of retinal and macular
degenerations caused by RPE dysfunction.
CR: R. Alper, None; A. Obolensky, None; M. Idelson, None; I. Hemo, None; R. Yaul,
None; B. Reubinoff, None; E. Banin, None.
Support: Yedidut Research Grant and the Israeli Ministry of Health
Purpose: Cell based therapies for the prevention of blindness have been shown to be
effective in animal models of human disease. We investigated conditions under which
highly-characterized human RPE cells derived from embryonic stem cell lines and
manufactured under Good Manufacturing Practice (GMP)-compliant conditions could
optimally rescue visual function in the RCS rat.
Methods: GMP-compliant hES-RPE cells from Advanced Cell Technology were injected
into the subretinal space of 22 day-old (P22) RCS rats in five different dosage groups:
5x103/eye (5K), 2x104/eye (20k), 5x104/eye (50K), 7.5x104/eye (75K) and 1x105/eye (100K).
With each group of animals, 3-4 eyes received injections of sham alone. The unoperated
eye provided further baseline data. All animals were maintained on oral cyclosporine
A (CyA) administered in the drinking water. The Optomotor test, a quick and repeatable
method of estimating rodent acuity, was conducted from P60-P240. Tectal recordings
were made at about P100 and P180. At the end of the testing, animals were sacrificed and
eyes were processed for histology.
Results: The OptoMotor test showed a dose dependent response: 50K, 75K and 100K
grafted animals performed significantly better than that of 5K, 20K, shams and untreated
animals, at P90; also, the visual acuity of RCS rats with 50K, 75K and 100K treatments
showed significant improvement (p<0.05) over control animals, at all time points.
Threshold recording from the superior colliculus showed that hES-RPE grafted animals
had lower visual thresholds compared with sham injected and untreated groups up to
P180. Histological analyses indicated that donor cells survived up to P210, with many
integrating into the host RPE cells layer. Substantial photoreceptor rescue was observed
in and beyond engrafted regions of the retina. There were no signs of tumor formation
or other unwanted pathology.
Conclusions: This study showed that a GMP-compliant human ES-derived cell line
injected into subretinal space of RCS rat can preserve photoreceptors and visual functions
in a dose-dependent manner. The mechanism underlying this morphological and
functional rescue may be neuroprotective factor-release or phagocytosis of photoreceptor
outer segments by donor cells. Our data suggest that hES-RPE may provide an effective
donor cell source to rescue photoreceptors in conditions such as AMD where RPE function
is compromised.
CR: B. Lu, None; S. Wang, None; S. Girman, None; P. Francis, None; C. Malcuit, E, E;
L. Vilner, E, E; L. Lemieux, E, E; R. Lanza, E, E; R. Lund, C, C.
Support: Advanced Cell Technology; Foundation Fighting Blindness and Lincy
Foundation.
Copyright 2008 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4280-4283
Wednesday, April 30, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 4276-4290 / A424-A438
440. AMD Surgical Therapy Organizing Section: RE Contributing Section: LE
4284 - A432
Photoreceptor-Like Differentiation of RPCInduced by RPE Cells
4285 - A433
Long Term Functional and Structural Outcomes of Macular Translocation and
Autologous Rpe Choroid Patch Graft for Treatment of Exudative Age-Related
Macular Degeneration
X. Deng1A, D. Zhu1A, C. Spee1B, S.J. Ryan1B,1C, D.R. Hinton1A,1C. APathology,
B
Ophthalmology, CDoheny Eye Institute, 1Keck School of Medicine of the
University of Southern California, Los Angeles, CA.
Purpose: The emerging field of retinal cell replacement therapy provides a promising
new approach for the treatment of retinal degeneration. The current study was focused
on evaluating conditions for in vitro induction of photoreceptor-like differentiation
of retinal progenitor cells (RPCs).
Method: 1). RPC isolation: retinas were dissected from human fetal eyes (18-20
gestational weeks) and digested with trypsin. The isolated RPCs were maintained
in RPC medium supplemented with 10% knockout replacement serum, N2 and B-27
supplement , EGF, bFGF, and IGF on fibronectin pre-coated culture dishes. 2). Induction
of RPC differentiation: Fetal retinal pigment epithelium (fRPE) and mouse embryonic
fibroblast (MEF) cells were treated with Mitomycin C (10 ug/ml), seeded on cell culture
dishes (8x105 cells/cm 2), and incubated overnight. The human RPCs were then seeded
either on top of fRPE or MEF cells, or on fibronectin coated dishes, cultured up to
7days, with culture medium changed every other day. 3). Immunofluorescent labeling:
RPCs on chamber slides were immuno-stained with anti-Nestin, Pax6 , Chx10 and
Rhodopsin antibody. The stained cells were observed and digitally imaged under
fluorescence microscopy.
Results: RPCs isolated from human fetal retina exhibited positive immunofluorescent
staining for three RPC markers; Nestin, Pax6 and Chx10. RPCs could temporally grow
on top of fRPE, MEF cells or on fibronectin coated dishes. However, after 7 days of
culture, only the RPCs on fRPE cells remained viable and healthy; the RPCs grown
on fibronectin coating showed degenerative changes; while most of the RPCs grown
on MEF cells died. There were more rhodopsin positive cells in the RPCs cultured on
RPE cells, than in cultures in which RPCs were grown on fibronectin.
Conclusion: RPCs are successfully isolated and cultured from 18-20 week gestation
human retinas. RPE cells provide support for RPC cell growth, and promote their
differentiation into photoreceptor-like cells in vitro.
CR: X. Deng, None; D. Zhu, None; C. Spee, None; S.J. Ryan, None; D.R. Hinton,
None.
Support: EY 01545 and EY 03040
F.K. Chen1A,2A, G.S. Uppal1A, R. MacLaren1A,2B, A. Tufail1B, G.W. Aylward1A, L. da Cruz1A.
Department of Vitreoretinal Surgery, BDepartment of Medical Retina, 1Moorfields
Eye Hospital, London, United Kingdom; ACellular Therapy, BMolecular Therapy,
2
UCL Institute of Ophthalmology, London, United Kingdom.
A
Purpose: To describe the two year outcomes of macular translocation with 360 degree
retinectomy (MT) and autologous RPE-choroid graft in patients with exudative agerelated macular degeneration (AMD)
Methods: We retrospectively reviewed the medical charts from the first 12 patients
who underwent MT (from May 2003 to April 2005) and the 12 patients who underwent
graft (from August 2004 to June 2005). Data on complications, visual acuity (VA),
microperimetry (MP) and fundus imaging (colour photographs, angiography, optical
coherence tomography, autofluorescence images) were reviewed.
Results: The mean age for the MT and graft groups were 74 and 80 respectively (p =
0.12). A Mann-Whitney U test revealed no significant difference in the baseline VA
between the MT group (Median = 0.90, n = 12) and the graft group (Median = 0.87,
n = 12), U = 58.5, p = 0.44. At 2 years, 5 and 2 of the 12 eyes each, that underwent MT
and graft, respectively, were seeing 20/100 or better. Friedman Test showed that
there was a statistically significant reduction in VA across the four time points in the
graft group (χ2 = 14.0, p = 0.03). The median VA deteriorated from baseline (median =
0.87) to 6 months (median = 1.41) and then stabilised between 1 (median = 1.50) and 2
years (median = 1.48). In contrast, the median VA improved slightly and maintained
for up to 2 years in the MT group (χ2 = 6.0, p = 0.11). Serial MP showed persistence of
retinal sensitivity and maintainance of fixation stability. Postoperative complications
within the first year after initial surgery included retinal detachment, submacular
haemorrhage, persistent or recurrent choroidal neovascular membrane (CNV), and
proliferative vitreoretinopathy and torsion. A total of 8 and 5 patients required
additional intraocular procedures for treatment of postoperative complications In
the MT and graft groups respectively. Complications arising during the second
or third year, include cystoid macular edema, recurrent CNV and alteration in the
autofluorescence pattern in the submacular region.
Conclusions: Overall VA is poor after graft despite re-establishment of photoreceptorRPE interface. The short and long term visual outcome of MT appears to be superior to
that of graft despite similar success rate in anatomical reconstitution or reconstruction
of the foveal-RPE-choroid interface.
CR: F.K. Chen, None; G.S. Uppal, None; R. MacLaren, None; A. Tufail, None; G.W.
Aylward, None; L. da Cruz, None.
Support: None
4286 - A434
Development and Characterization of Adult Retinal Explant Organotypic
Tissue Culture as an in vitro Model for Intravitreal Stem Cell Transplantation
4287 - A435
Three-Dimensional Optical Coherence Tomography Imaging of Retinal Sheet
Implants in Live Rats
T.V. Johnson, K.R. Martin. Brain Repair Centre, Cambridge University, Cambridge,
United Kingdom.
M.J. Seiler1A, R. Aramant2,1A, B. Rao1B,1C, L. Yu1B, Q. Wang1B, S. Pham1B, Z. Chen1B,
F. Yan1A, H.K. Keirstead1A. AReeve-Irvine Res Ctr., Sue and Bill Gross Stem Cell
Research Ctr., Dept. Anatomy & Neurobiol., BDept. Biomed. Engineering, Beckman
Laser Inst., CDept. Electrical Engineering & Computer Science, 1UC Irvine, Irvine,
CA; 2Dept. Anatomy & Neurobiol., Univ. Louisville, Louisville, KY.
Purpose: Transplantation of stem/progenitor cells to treat retinal neurodegenerative
disease is a subject of intense investigation. Current therapeutic efforts are limited
by low numbers of cells integrating into the retina and suboptimal control over the
differentiation and behaviour of grafts. To facilitate investigation into novel methods
of improving retinal stem cell therapy, we developed a retinal explant culture system
using adult rats.
Methods: Retinas were explanted from 8-12 week old Sprague Dawley rats retinal ganglion
cell (RGC) side up on Millipore filters in B27/N2-supplemented serum-free media or in
media containing 25% normal horse serum (NHS) for up to 17 days. Tissue viability was
assessed at various time points by gross morphology, propidium iodide (PI) uptake,
quantification of cell survival, activated caspase-3 expression, and protein expression
patterns. To model intravitreal cell transplantation, 1-2x103 human Müller progenitor
cells (hMIO-M1) in 2µL of media were placed on explants. Explant morphology and
immunohistochemistry were compared to sectioned whole eyes with or without prior
intravitreal hMIO-M1 transplantation.
Results: Explants cultured in B27/N2 media were viable through 17 days as evidenced
by PI exclusion, static cell densities, consistently low caspase-3 expression and little
morphological change. In contrast, NHS media was associated with obvious tissue
degradation beginning peripherally and encompassing 73±4% (mean ± SEM) of the
tissue by day 17; greater and more diffuse PI uptake; significant cell loss over time,
especially from the RGC layer (77±5 on day 3 vs. 14±2 cells/mm on day 14, p<0.01);
and a temporal increase in active caspase-3+ cells (4±1% on day 3 vs. 19±5% on day 17
in the RGC layer, p<0.01). Explants in B27/N2 media were strongly immunoreactive
for β-III-tubulin, neurofilament, NeuN, Brn3a, and Thy-1 as well as GFAP, vimentin,
nestin, and glutamine synthetase in the inner retina, whereas expression was weak for
NHS media and decreased with time. Seven days after transplantation, glial reactivity
as assessed by GFAP expression was highly upregulated in explants and control eyes.
Some grafted cells migrated into the retina, but the majority remained outside of the
inner limiting membrane.
Conclusions: Retinal explants cultured in B27/N2 media are viable for at least 2 weeks
and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to
integrate. This system will be useful for investigating methods to enhance retinal stem
cell therapy by providing a manipulatable in vitro model.
CR: T.V. Johnson, None; K.R. Martin, None.
Support: TVJ: Gates-Cambridge Scholarship, NIH-GPP Studentship, Fight for Sight
Summer Student Fellowship; KRM: GSK Clinician-Scientist Award, Glaucoma Research
Foundation Grant
Purpose: To obtain three-dimensional images from retinal transplants in live animals
and evaluate the placement and structural quality of the transplants.
Methods: Donor retinal sheets were isolated from E19 fetuses of transgenic rats
expressing human alkaline phosphatase (hPAP) as a marker, and transplanted to the
subretinal space of 24 - 56 d old S334ter-3 rat recipients with fast retinal degeneration.
Forty-one rats were imaged 6 - 67 d after transplant surgery, at the age of 36 to 107
d, using a Fourier domain optical coherence tomography (FDOCT) system, with an
axial resolution of 3.5 micron. The CCD A-line integration time was set at 200 µs for
better visualization of degenerated retina. After targeting the transplant area, 198
consecutive slices were scanned. From these scans, 3D-projection images and movies
of the retinal transplant area were computed.
Results: Transplants could easily be identified. OCT scans also showed whether
the transplants had been correctly placed into the subretinal space, and whether
there had been damage to the RPE or choroid during implantation. OCT indicated
the laminar structure of the implants. Three-dimensional projections showed the
transplant position in the retina in relation to the optic disc. Transplants were identified
histologically by hPAP staining.
Conclusions: Optical Coherence Tomography is an excellent tool to image retinal
layers in a live rat. This is important to assess the transplant success early after
surgery. This procedure helps to evaluate the placement and layering of the implants
in the living eye.
CR: M.J. Seiler, Ocular Transplantation LLC, P; R. Aramant, Ocular Transplantation
LLC, P; Ocular Transplantation LLC, E; B. Rao, None; L. Yu, None; Q. Wang, None; S.
Pham, None; Z. Chen, None; F. Yan, None; H.K. Keirstead, None.
Support: Lincy Foundation, NIH (EB-00293, NCI-91717, RR-01192), Air Force Office
of Science Research (FA9550-04-1-0101), Institutional support from BLI and Beckman
Medical Clinic.
Copyright 2008 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4284-4287
Wednesday, April 30, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 4276-4290 / A424-A438
440. AMD Surgical Therapy Organizing Section: RE Contributing Section: LE
4288 - A436
Long-Term Changes in Rat Eyes After Intravitreal Transplantation of Cultured
Müller Cells
M. Nakatani, M. Hosoi, Y. Shinohara, C. Taki, M. Hirabayashi, S. Nishimura.
Bioengineering Institute, Nidek Co., Ltd., Aichi, Japan.
Purpose: Intravitreally-transplanted Müller cells are likely to protect retinal ganglion
cells from death induced by optic nerve (ON) injury (Hosoi, ARVO 2007). This study
was to morphologically examine the fate of transplanted Müller cells as well as their
effects on the host eye.
Methods: Müller cells were isolated from retinas of 14-day-old Wistar/ST rats. After
being cultivated and labeled with the lipophilic fluorescent dye (PKH26), approximately
4 × 105 cells were injected into the vitreous bodies of syngeneic adult rats bilaterally.
Three days after the transplant, ON injuries were made in the right eyes by applying a
vessel clip. Rats were euthanized at 2, 4, 13 and 26 weeks after the clamping. Paraffinembedded and frozen sections were processed for hisotopathological examination
and immunohistochemistry. Retinal flat-mounts were also prepared and examined
for the distribution of transplanted cells.
Results: The majority of transplanted cells existed in the form of clots attached on
the lens posterior capsule or into the vitreous cavity, while some cells were seen on
the retinal surface. The cells on the lens posterior capsule formed a fibrocellular
membrane by 2 weeks after transplantation. The cells on the retinal surface distributed
over more than half of the area, frequently accumulating in the optic papilla and
along major blood vessels. These cells survived for 26 weeks although they gradually
decreased in their numbers. Localized retinal detachment occurred in 2 eyes (12.5%)
without ON injury and in 6 eyes (37.5%) with ON injury between 2 and 26 weeks after
transplantation. Immunohistochemistry revealed enhanced expression of the glial
fibrillary acidic protein (GFAP) from 2 to 26 weeks after transplantation.
Conclusions: Transplanted Müller cells remained alive in the vitreous cavity or on
the retinal surface, and activated the host retina at least for 6 months. Phenotypic
changes of the cells into fibrous membranes were found on the lens capsule as well
as along vitreous fibrils. Such changes not only cause lens opacity, but may also be
inducing tractional retinal detachments.
CR: M. Nakatani, None; M. Hosoi, None; Y. Shinohara, None; C. Taki, None; M.
Hirabayashi, None; S. Nishimura, None.
Support: None
4289 - A437
Autologous Retinal Pigment Epithelium and Choroid Transplantation With
180 Degrees Peripherical Retinotomy in Patients With Exudative Age-Related
Macular Degeneration: Short Term Follow-Sp and Anatomical Outcome
M.G. Cereda1,2, B. Parolini1, G. Staurenghi2, G. Pertile1. 1Department of Ophthalmology,
Sacro Cuore Hospital of Negrar, Verona, Italy; 2Clinical Science Luigi Sacco, Eye
Clinic, Luigi Sacco Hospital, University of Milan, Milano, Italy.
Purpose: to evaluate the feasibility of transplanting autologous retinal pigment
epithelium (RPE) cells, choriocapillaris and choroid by a 180° peripheral temporal
retinotomy after the removal of a subfoveal choroidal neovascularization (CNV) in
patients with age-related macular degeneration.
Methods: a prospective, observational case series study on 8 patients was developed
in order to investigate the topic with a follow-up of 4 to 12 months. All patients had
CNV, 3 with vitreous haemorrage, 4 with wide subretinal haemorrhage and 1 with
serous-haemorrhagic retinal pigment epithelium detachment. Preoperative visual
acuity (VA) ranged from Light Perception to logMar 1.3. After the induced posterior
and temporal retinal detachment, the peripheral 180° temporal retinotomy and the
extraction of the CNV, an autologous full-thickness patch of choroid, choriocapillaris,
Bruch’s membrane and RPE was harvested from the mid-periphery and, after the
injection of an heavy liquid, repositioned beneath the macula. All patients underwent
a complete ophthalmic examination, Optical Coherence Tomography, Scanning Laser
Ophthalmoscopy autofluorescence, dynamic Indocyanine Green Angiography (ICGA)
and Fluorescence Angiography.
Results: the full thickness patch appeared flat, brown and well centered under the
fovea in seven patients, one had a wrinkled nasal margin. Post operative vision
ranged from LogMar 2.6 to LogMar 0.4; VA increased in 7 patients and remained
stable in 1. Revascularization was visible on ICGA in all the patients. In one patient
revascularization was absent in the nasal area of the patch after 4 months follow-up.
Normal RPE autofluorescence was present over the patch in all the patients. No intra
or post operative complications occurred.
Conclusions: the transplantation of a full thickness patch of choroid, choriocapillaris,
Bruch’s membrane and RPE under the macula after a peripheral temporal 180°
retinotomy and the extraction of the CNV is feasible and may result in a surviving
and functioning graft for more than 6 months. A longer follow-up is necessary to
evaluate the efficacy and safety of this treatment.
CR: M.G. Cereda, None; B. Parolini, None; G. Staurenghi, None; G. Pertile,
None.
Support: None
4290 - A438
Cytoprotective Effects of a Blue-Light-Absorbing IOL on Human RPE by
Reduced Phototoxic Decrease of Bcl-2
M. Kernt, U. Welge-Lussen, R. Liegl, A.S. Neubauer, A. Kampik. Ophtalmology,
Augenklinik der Universitat Muenchen, Gruenwald, Germany.
Background: Light-induced phototoxicity is implicated to trigger apoptotic cell loss
in the retinal pigment epithelium (RPE) and to support development of age related
macular degeneration (AMD). Recently, to prevent the retina from blue-light damage
in pseudophacia, blue-light-absorbing intraocular lenses (IOL) have been introduced.
This study compares possible protective effects of the blue-light-absorbing ALCON
SN60AT IOL to the untinted, UV-absorbing SA60AT IOL regarding light-induced
stress on human RPE.
Methods: Primary human RPE cells were exposed to white light and either a SN60AT
or SA60AT IOL was placed in the light beam. After 10 to 60 minutes of irradiation, the
viability of the cells was determined by a colorimetric test (MTT), and expression of
VEGF-A and Bcl-2 and their mRNA were determined by RT-PCR and Western-Blot
analysis.
Results: Without any IOL, white-light exposure decreased cell viability compared to
the nonirradiated control in a time-of-irradiation-dependent manner. Light-induced
cell death was significantly reduced by both, the SN60AT and SA60AT IOL. The
blue-light filtering of the SN60AT further significantly attenuated light-induced
cell damage as compared to the SA60AT IOL. RT-PCR and Western-Blot analysis
yielded a significant, time-of-irradiation-dependent decrease of Bcl-2 and an increase
of VEGF-A. In the presence of an IOL, Bcl-2 levels decreased and VEGF-A levels
increased significant less. In addition, this decrease of Bcl-2 and increase of VEGF-A
was significantly less for the SN60AT IOL compared to the SA60AT IOL.
Conclusion: Our findings demonstrate, that both, UV-filtering and blue-light-absorbing
IOLs reduce light-induced RPE-damage. The blue-light-absorbing IOL further reduced
damage compared to the conventional IOL, which supports the hypothesis of possibly
also preventing retinal damage in clinical use.
CR: M. Kernt, None; U. Welge-Lussen, None; R. Liegl, None; A.S. Neubauer, None; A.
Kampik, None.
Support: None
Copyright 2008 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4288-4290