advances in
REGENERATIVE BIOLOGY
æ
MINI REVIEW
Adult human RPE for transplantation: renewing an
old promise
Timothy A. Blenkinsop*
Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Icahn School
of Medicine at Mount Sinai, New York, NY, USA
Received: 31 December 2014; Revised: 4 March 2015; Accepted: 8 March 2015; Published: 6 April 2015
Abstract
In context
Considering the incidence of retinal pigment epithelium
(RPE)-related blinding disease will grow to 200 million
globally by 2020, the impact of restoring vision by successfully
replacing failing or dying RPE is great. In spite of fervent
efforts to use primary RPE as a source for transplantation for
over 30 years, a clinical therapy has yet to be developed. Due
to the progress of pluripotent stem cell technologies and
development of RPE differentiation protocols, primary human RPE culture has largely been set aside as a source of RPE
for transplantation, as human embryonic stem cell (hESC)and induced pluripotent stem cell (hiPSC)-derived RPE have
become the current popular source for transplantation. Recently, a series of seminal advances in human primary RPE
culture has renewed an interest in their potential as a source
for RPE transplantation. Primary RPE are better studied and
understood than hESC/hiPSC-derived RPE, have an inherent
lower risk of tumor formation, and can be Major Histocompatibility Complex (MHC) donor-matched, making them valuable candidates alongside pluripotent stem cells as sources
for cell transplantation therapy for RPE-related eye diseases.
Keywords: adult stem cell;
transplantation; eye disease
retinal
pigment
epithelium;
cell
Some of the most prevalent blinding diseases, including Agerelated Macular Degeneration, Stargardt’s Disease, Retinitis
Pigmentosa and others, affect a single epithelial layer in the
back of the eye, called the retinal pigment epithelium (RPE).
For over the past 40 years, much hope has rested in using
adult RPE, for example isolated from cadaver donors,
for transplantation, to replace the diseased RPE in affected
patients. Critical barriers to this objective are 1. being able to
isolate and grow RPE that maintain their physiological and
morphological characteristics in vitro and 2. assure successful engraftment and survival of the transplanted cells. What
we observed was that often, once dissected, RPE isolated
from cadaver donor eyes would change their physiology and
not maintain their RPE functions when cultured in vitro.
Here we summarize new advances in using adult RPE, which
have renewed their promise in treating RPE-related eye
diseases.
*Correspondence to: Timothy A. Blenkinsop, Department of Developmental and Regenerative Biology, Black Family
Stem Cell Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Ave., New York, NY 10029, USA,
Email: timothy.blenkinsop@mssm.edu
he retinal pigment epithelium (RPE) is a single
layer of cells, which performs many functions
necessary for healthy phototransduction (1) and
any dysfunction in these roles leads to vision loss. Cell
transplantation of healthy RPE to replace dystrophic
or lost RPE has been considered a potential therapy to
preserve or even restore lost vision. Considering the
number of individuals with RPE-related blinding disease
to be over 200 million worldwide by 2020 (2), obtaining
sufficient RPE for such a demand has been a challenge on
which many in the RPE field are focused. Here, we cover
some of the roadblocks, surgical successes, and recent
T
developments that renew the sense that we are close to
providing a transplantation therapy for patients suffering
from RPE-related diseases.
Much hope rests on the idea that a simple ‘RPE-only’
transplantation can be developed, a method that can be
scaled up to threat such a large population of patients.
The foundation of the hope rests on a handful of surgical
successes. Macular translocation is the surgical rotation of
the retina so that the retina is placed on a healthy RPE
layer. While the success rate is variable due to the complexity of the surgery, when there are no complications,
macular translocation has demonstrated dramatically
Advances in Regenerative Biology 2015. # 2015 Timothy A. Blenkinsop. This is an Open Access article distributed under the terms of the Creative Commons
Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or
format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Citation: Advances in Regenerative Biology 2015, 2: 27144 - http://dx.doi.org/10.3402/arb.v2.27144
1
(page number not for citation purpose)
Timothy A. Blenkinsop
improved vision (3, 4). Similarly, a peripheral RPEchoroid patch translocation underneath the macula also
improves vision, yet the risks of complications are high (5).
As a result, only a handful of retinal surgeons attempt
either of these surgeries and few patients receive their
benefit.
Current state of the RPE transplantation
Transplanting cells as a therapy for human disease is
relatively new and limited primarily to blood-based diseases (6, 7), skin (8), and cornea (9), though much hope
rides on its potential to be effective in treating diseases
throughout the human body. RPE transplantation for
RPE-related diseases is at the forefront of this movement,
because these cells and their location possess many attractive attributes when developing a cell therapy. The RPE
is a single layer and is therefore considered to be one of the
more easily transplantable tissues, because elaborate or
long-distant connections do not need to be re-established.
The eye has a unique feature in providing a window, the
cornea, through which progress of the transplanted tissue
can be easily monitored. RPE are pigmented and distinguishable from the transparent retina, particularly in the
regions where native RPE is lost. Sophisticated ophthalmological surgical techniques, imaging devices and visual
acuity tests are additional benefits, making the eye the
ideal location to pioneer the development and optimization of cell transplantation therapy.
Alongside the struggles of primary RPE culture was the
development of human embryonic stem cell (hESC) (10),
retinal neurospheres (11), induced pluripotent stem cell
(hiPSC) cultures (12), and differentiation methods towards
the RPE lineage (13, 14). These cell sources hold the promise of unlimited supply and, in the case of hiPSC, autologous transplantation potential. As a result, attention to
and development of primary human RPE as a source for
transplantation therapy have largely been set aside.
Many groups are moving swiftly into the clinic to test
whether these seemingly unlimited sources of cells will
be the next clinical revolution. A clinical trial, funded by
Ocata Therapeutics, Inc. (formerly Advanced Cell Technology), is ongoing, where hESC-derived RPE are injected
into the subretinal space as a single cell suspension. Preliminary reports indicate the treatment so far is safe, and
has led to an improvement in vision in some patients (15,
16). The London Project to Cure Blindness led by Dr. Pete
Coffey in collaboration with Pfizer plans to use hESCderived RPE attached to a porous polyester scaffold to
replace lost RPE (http://www.theguardian.com/science/
2009/jan/30/stemcells-genetics).
Human iPSC-derived RPE are not far behind. A clinical
trial is in its early stage at the RIKEN Institute in Kobe,
Japan, led by stem cell biologist Dr. Masayo Takahashi,
using iPSCs to establish patient-specific cells for transplantation to reduce immune rejection. According to a
2
(page number not for citation purpose)
press release from the RIKEN Institute (http://www.rikenibri.jp/AMD/english/index.html), a sheet of hiPSC-derived
RPE was transplanted underneath a patient’s existing
retina as an informal clinical safety test. An exercise of
caution is warranted as there have been some reports of
immune attack on genetically matched iPSCs (1720),
tumorigenicity (21) and loss of RPE characteristics in
later passages that reduce their expansion potential and
increases muscle contraction markers, a risk factor for
Proliferative Vitreoretinopathy (22). On the other hand,
a safety study demonstrated that transplantation of 1.
5104 iPSC-derived RPE in a collagen-lined sheet elicited
no tumor formation after 15 months in an immunodeficient mouse (23).
Primary RPE culture as a source for
transplantation
The first culture of RPE cells was done almost 100 years
ago with the in vitro cultivation of the embryonic optic cup
from 72-h chick embryos, demonstrating their continual
development in the absence of neural connections or blood
supply (24). Not until 1973 would a method of culturing
RPE as a monolayer be developed (25). Clinical implications were clearly in the authors’ mind as they note that
these cultures may ‘form valuable substrates for pharmaceutical research’. Interestingly, the observation of epithelial morphology loss was also acknowledged, which is still
a dominant factor delaying RPE transplantation therapy.
The year 2015 will be the 30th anniversary of the first
publication on the transplantation of RPE (26): reestablishment of a pseudo-monolayer was observed within
24 h of transplant in the owl monkey. There was, however,
indication of an inflammatory response as early as a few
hours after transplantation, foreshadowing what many
since have struggled to overcome (2733).
For over 30 years, focus has been directed towards
primary adult human RPE cultures as a promising source
of RPE transplantation. Adult RPE cultured from human
cadaver donors have been examined but their transformation from a homogenous cobblestone monolayer to a mix
of distinct morphologies in vitro was observed (34). Cultured RPE undergo an epithelial to mesenchymal transition (EMT), in which the cells lose their epithelial features,
including tight junctional complexes, polarized membranes
and shape, and gain mesenchymal characteristics, such
as migration, morphological elongation and contractilelike properties (35, 36). The failure of attachment of cultured adult human RPE onto aged Bruch’s membrane
has been observed and can be a problem in transplantation, but can be reversed by Extracellular matrix (ECM)
reconstitution (37).
More recently, methods in preventing EMT in primary
cultures have been established. Porcine cultures have been
shown to reverse initial EMT progress and were able
to re-establish a cobblestone monolayer using Ca2 as a
Citation: Advances in Regenerative Biology 2015, 2: 27144 - http://dx.doi.org/10.3402/arb.v2.27144
RPE for transplantation therapy
trigger (38). Cell culture media allowing a much more
homogenous culture of fetal RPE was also developed (39).
An improved culture method and media were described,
capable of maintaining fetal human RPE monolayers that
displayed native RPE electrophysiology, protein polarization, apical-to-basal fluid transport and more (40). This
was the most extensive demonstration of native RPE
physiology from a human RPE culture and introduced
human fetal RPE as a promising candidate for RPE transplantation. Nonetheless, limited tissue resources, potential ethical or political resistance reduced enthusiasm for
this cell as a source for RPE transplantation.
The renewed promise of primary RPE
Deceased human donors have been considered a possible
source of RPE for transplantation for over 30 years. Unfortunately, RPE freshly isolated from human donors do
not reattach to the Bruch’s membrane following detachment from the original host Bruch’s membrane (41). RPE
lose the integrins necessary for reattachment and cannot
reproduce them quick enough to reestablish a functional
relationship with the choriocapillaris before undergoing
apoptosis. To reverse this, perhaps establishment of novel
culture conditions in which cells will be stimulated, possibly by a cytokine provided in the media, will allow RPE
to re-attach to the Bruch’s membrane, survive and maintain RPE identity before EMT is irreversibly completed.
More recently, a stem cell has been identified in the
adult human RPE layer (RPESC), which can divide to reestablish an RPE epithelial monolayer (42). A novel
culture method has also been developed, which allows
maintenance of the epithelial physiology of cultured adult
human RPE without undergoing EMT (43). This method
promotes cellcell contact inhibition while fostering
moderate levels of proliferation, therefore maintaining a
delicate balance between expansion and dedifferentiation.
5106 RPE cells can be obtained from one donor. In
order to cover the macula, some estimates have shown
that as low as 5104 RPE are sufficient, therefore up
to 100 patients could be treated with RPE cells isolated
from one single donor. Moreover, these cultures can be
expanded 100-fold, producing up to 5 108 cobblestone
RPE from one donor, sufficient to treat up to 10,000
patients. Once transplanted as a sheet attached on a
polyester porous scaffold in a rabbit model, RPE not only
survived for more than 1 month, but were also shown to
remain a polarized epithelial monolayer (33). Recently
a contract was granted by New York State NYSTEM
agency to Dr. Sally Temple to develop a cell transplantation therapy using primary adult human RPE (http://
stemcell.ny.gov/nystem-consortia). Taken together, these
developments renew the adult human RPE as a cell source
for cell transplantation therapy and may be considered
alongside hESC- and hiPSC-derived RPE as promising
technology for patients with RPE-related diseases.
Conflict of interest and funding
The authors have not received any funding or benefits from
industry or elsewhere to conduct this study.
References
1. Strauss O. The retinal pigment epithelium in visual function.
Physiol Rev. 2005; 85: 84581.
2. Wong WL, Su X, Li X, Cheung CM, Klein R, Cheng CY, et al.
Global prevalence of age-related macular degeneration and
disease burden projection for 2020 and 2040: a systematic review
and meta-analysis. Lancet Global Health. 2014; 2: e10616.
3. Machemer R, Steinhorst UH. Retinal separation, retinotomy,
and macular relocation: II. A surgical approach for age-related
macular degeneration? Graefes Arch Clin Exp Ophthalmol.
1993; 231: 63541.
4. Seaber JH, Machemer R. Adaptation to monocular torsion
after macular translocation. Graefes Arch Clin Exp Ophthalmol. 1997; 235: 7681.
5. Stanga PE, Kychenthal A, Fitzke FW, Halfyard AS, Chan R,
Bird AC, et al. Retinal pigment epithelium translocation after
choroidal neovascular membrane removal in age-related macular degeneration. Ophthalmology. 2002; 109: 14928.
6. Blau O, Blau IW. Some aspects of allogeneic stem cell transplantation in patients with myelodysplastic syndrome: advances
and controversy. Stem Cells Cloning. 2014; 7: 1018.
7. Garcia IN. Role of hematopoietic stem cell transplantation in
multiple myeloma. Clin Lymphoma Myeloma Leukemia. 2014;
15: 8691.
8. Billingham RE, Reynolds J. Transplantation studies on sheets of
pure epidermal epithelium and on epidermal cell suspensions.
Br J Plast Surg. 1952; 5: 2536.
9. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda
R, De Luca M. Long-term restoration of damaged corneal
surfaces with autologous cultivated corneal epithelium. Lancet.
1997; 349: 9903.
10. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA,
Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines
derived from human blastocysts. Science. 1998; 282: 11457.
11. Bertolotti E, Neri A, Camparini M, Macaluso C, Marigo V.
Stem cells as source for retinal pigment epithelium transplantation. Prog Retin Eye Res. 2014; 42: 13044.
12. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T,
Tomoda K, et al. Induction of pluripotent stem cells from adult
human fibroblasts by defined factors. Cell. 2007; 131: 86172.
13. Klimanskaya I, Hipp J, Rezai KA, West M, Atala A, Lanza R.
Derivation and comparative assessment of retinal pigment
epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells. 2004; 6: 21745.
14. Hirami Y, Osakada F, Takahashi K, Okita K, Yamanaka S,
Ikeda H, et al. Generation of retinal cells from mouse and
human induced pluripotent stem cells. Neurosci Lett. 2009; 458:
12631.
15. Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V,
Pan CK, Ostrick RM, et al. Embryonic stem cell trials for
macular degeneration: a preliminary report. Lancet. 2012; 379:
71320.
16. Schwartz SD, Regillo CD, Lam BL, Eliott D, Rosenfeld PJ,
Gregori NZ, et al. Human embryonic stem cell-derived retinal
pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two
open-label phase 1/2 studies. Lancet. 2015; 385: 50916.
17. Dressel R, Nolte J, Elsner L, Novota P, Guan K, StreckfussBomeke K, et al. Pluripotent stem cells are highly susceptible
Citation: Advances in Regenerative Biology 2015, 2: 27144 - http://dx.doi.org/10.3402/arb.v2.27144
3
(page number not for citation purpose)
Timothy A. Blenkinsop
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
targets for syngeneic, allogeneic, and xenogeneic natural killer
cells. FASEB J. 2010; 24: 216477.
Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced
pluripotent stem cells. Nature. 2011; 474: 21215.
Araki R, Uda M, Hoki Y, Sunayama M, Nakamura M, Ando S,
et al. Negligible immunogenicity of terminally differentiated
cells derived from induced pluripotent or embryonic stem cells.
Nature. 2013; 494: 1004.
Cao J, Li X, Lu X, Zhang C, Yu H, Zhao T. Cells derived from
iPSC can be immunogenic yes or no? Protein Cell. 2014; 5:
13.
Doi D, Morizane A, Kikuchi T, Onoe H, Hayashi T, Kawasaki
T, et al. Prolonged maturation culture favors a reduction in the
tumorigenicity and the dopaminergic function of human ESCderived neural cells in a primate model of Parkinson’s disease.
Stem Cells. 2012; 30: 93545.
Singh R, Phillips MJ, Kuai D, Meyer J, Martin JM, Smith MA,
et al. Functional analysis of serially expanded human iPS cellderived RPE cultures. Invest Ophthalmol Vis Sci. 2013; 54:
676778.
Kanemura H, Go MJ, Shikamura M, Nishishita N, Sakai N,
Kamao H, et al. Tumorigenicity studies of induced pluripotent
stem cell (iPSC)-derived retinal pigment epithelium (RPE) for
the treatment of age-related macular degeneration. PLoS One.
2014; 9: e85336.
Strangeways TSP, Fell HB. Experimental studies on the
differentiation of embryonic tissues growing in vivo and in
vitro. II. The development of the isolated early embryonic eye
of the fowl when cultivated in vitro. Proc Roy Soc Lond. 1926;
100: 27383.
Mannagh J, Arya DV, Irvine AR Jr. Tissue culture of human
retinal pigment epithelium. Invest Ophthalmol. 1973; 12: 5264.
Gouras P, Flood MT, Kjedbye H, Bilek MK, Eggers H. Transplantation of cultured human retinal epithelium to Bruch’s
membrane of the owl monkey’s eye. Curr Eye Res. 1985; 4:
25365.
Sheng Y, Gouras P, Cao H, Berglin L, Kjeldbye H, Lopez R,
et al. Patch transplants of human fetal retinal pigment epithelium in rabbit and monkey retina. Invest Ophthalmol Vis Sci.
1995; 36: 38190.
Castellarin AA, Sugino IK, Vargas JA, Parolini B, Lui GM,
Zarbin MA. In vitro transplantation of fetal human retinal
pigment epithelial cells onto human cadaver Bruch’s membrane.
Exp Eye Res. 1998; 66: 4967.
Algvere PV, Gouras P, Dafgard Kopp E. Long-term outcome of
RPE allografts in non-immunosuppressed patients with AMD.
Eur J Ophthalmol. 1999; 9: 21730.
4
(page number not for citation purpose)
30. Crafoord S, Algvere PV, Kopp ED, Seregard S. Cyclosporine
treatment of RPE allografts in the rabbit subretinal space. Acta
Ophthalmol Scand. 2000; 78: 1229.
31. Gullapalli VK, Sugino IK, Van Patten Y, Shah S, Zarbin MA.
Impaired RPE survival on aged submacular human Bruch’s
membrane. Exp Eye Res. 2005; 80: 23548.
32. Tezel TH, Del Priore LV, Berger AS, Kaplan HJ. Adult retinal
pigment epithelial transplantation in exudative age-related
macular degeneration. Am J Ophthalmol. 2007; 143: 58495.
33. Stanzel BV, Liu Z, Somboonthanakij S, Wongsawad W, Brinken
R, Eter N, et al. Human RPE stem cells grown into polarized
RPE monolayers on a polyester matrix are maintained after
grafting into rabbit subretinal space. Stem Cell Rep. 2014; 2:
6477.
34. Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM.
ARPE-19, a human retinal pigment epithelial cell line with
differentiated properties. Exp Eye Res. 1996; 62: 15569.
35. Burke JM, Skumatz CM, Irving PE, McKay BS. Phenotypic
heterogeneity of retinal pigment epithelial cells in vitro and
in situ. Exp Eye Res. 1996; 62: 6373.
36. Burke JM. Epithelial phenotype and the RPE: is the answer
blowing in the Wnt? Prog Retin Eye Res. 2008; 27: 57995.
37. Tezel TH, Del Priore LV, Kaplan HJ. Reengineering of aged
Bruch’s membrane to enhance retinal pigment epithelium repopulation. Invest Ophthalmol Vis Sci. 2004; 45: 333748.
38. Rak DJ, Hardy KM, Jaffe GJ, McKay BS. Ca-switch
induction of RPE differentiation. Exp Eye Res. 2006; 82:
64856.
39. Hu J, Bok D. A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers. Mol Vis. 2001; 7: 1419.
40. Maminishkis A, Chen S, Jalickee S, Banzon T, Shi G, Wang FE,
et al. Confluent monolayers of cultured human fetal retinal
pigment epithelium exhibit morphology and physiology of
native tissue. Invest Ophthalmol Vis Sci. 2006; 47: 361224.
41. Tsukahara I, Ninomiya S, Castellarin A, Yagi F, Sugino IK,
Zarbin MA. Early attachment of uncultured retinal pigment
epithelium from aged donors onto Bruch’s membrane explants.
Exp Eye Res. 2002; 74: 25566.
42. Salero E, Blenkinsop TA, Corneo B, Harris A, Rabin D, Stern
JH, et al. Adult human RPE can be activated into a multipotent
stem cell that produces mesenchymal derivatives. Cell Stem Cell.
2012; 10: 8895.
43. Blenkinsop TA, Salero E, Stern JH, Temple S. The culture and
maintenance of functional retinal pigment epithelial monolayers
from adult human eye. Methods Mol Biol. 2013; 945: 4565.
Citation: Advances in Regenerative Biology 2015, 2: 27144 - http://dx.doi.org/10.3402/arb.v2.27144