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Progress in Retinal and Eye Research xxx (2014) 1e21
Contents lists available at ScienceDirect
Progress in Retinal and Eye Research
journal homepage: www.elsevier.com/locate/prer
Patient-specific induced pluripotent stem cells (iPSCs) for the study
and treatment of retinal degenerative diseases
Luke A. Wiley a, 1, 2, Erin R. Burnight a, 1, 2, Allison E. Songstad a, 1, 2, Arlene V. Drack a, 1,
Robert F. Mullins a, 1, Edwin M. Stone a, b, 1, Budd A. Tucker a, *, 1
a
b
Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA
Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2014
Received in revised form
15 October 2014
Accepted 16 October 2014
Available online xxx
Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is
one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular
degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world,
collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades,
scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with
retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach.
In this review we will discuss the use of the induced pluripotent stem cell technology to model and
develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will
focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement.
© 2014 Published by Elsevier Ltd.
Keywords:
Induced pluripotent stem cells
Retinal degeneration
Gene therapy
CRISPR-based genome editing
Cell transplantation
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1.
Cellular potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.2.
Origin of isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Induced pluripotent stem cells (iPSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disease modeling using iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Mendelian retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
MAK-associated retinitis pigmentosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Usher syndrome type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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List of abbreviations: AMD, age-related macular degeneration; RPE, retinal pigmented epithelium; ESC, embryonic stem cell; ASC, adult stem cell; iPSC, induced
pluripotent stem cell; RP, retinitis pigmentosa; AAV, adeno-associated virus; LCA,
Leber congenital amaurosis; TALEN, transcription activator-like effector nuclease;
CRISPR, clustered regulatory interspaced short palindromic repeat; ZFN, zinc finger
nuclease; sgRNA, small guide RNA; DSB, double-strand break; NHEJ, non-homologous end joining; InDel, insertion or deletion; HDR, homology-directed repair; RPC,
retinal progenitor cell; ACAID, anterior chamber-associated immune deviation.
* Corresponding author. Stephen A. Wynn Institute for Vision Research, Carver
College of Medicine, University of Iowa, Department of Ophthalmology and Visual
Sciences, 375 Newton Road, Iowa City, IA 52242, USA. Tel.: þ1 319 355 7242.
E-mail address: [email protected] (B.A. Tucker).
1
Percentage of work contributed by each author in the production of the
manuscript is as follows: Luke A. Wiley: 20%; Erin R. Burnight: 20%; Allison E.
Songstad: 20%; Arlene V. Drack: 7.5%; Robert F. Mullins: 7.5%; Edwin M. Stone: 7.5%;
Budd A. Tucker: 17.5%.
2
These authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
1350-9462/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
2
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
3.
4.
5.
2.4.
CEP290-associated Leber congenital amaurosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Juvenile neuronal ceroid lipofuscinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.
Best disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.
Using iPSCs to model AMD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gene augmentation and cell replacement treatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
iPSCs and gene augmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
iPSCs and genome editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
iPSCs for testing of therapeutic efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
iPSCs and drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.
Retinal transplantation and cellular replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.
Retinal immune landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Heritable retinal degenerative diseases, which include Mendelian disorders such as retinitis pigmentosa, Stargardt disease and
Leber congenital amaurosis, as well as the more complex and
heterogeneous disease, age-related macular degeneration (AMD),
are collectively the leading cause of incurable blindness in the
developed world (Huang et al., 2011). For years, ophthalmologists
and vision scientists have dreamed of restoring vision to those that
have lost this precious sense. Fortunately, despite widespread
death of the photoreceptors, retinal pigmented epithelium (RPE)
and choroidal endothelial cells e which comprise the outer retina–
the inner retina, which consists of ganglion cells, bipolar cells,
amacrine cells, horizontal cells, and Müller glia is largely spared in
these diseases (Huang et al., 2011; Mullins et al., 2009, 2012).
Preservation of the inner neural retina presents an opportunistic
environment in which preservation and replacement of these outer
retinal cell populations could maintain and restore visual function.
Over the past decade, studies employing stem cells for disease
modeling and treatment of incurable diseases have gained momentum in the field of regenerative medicine. The groundwork of
the stem cell field goes back more than 50 years when Sir John
Gurdon demonstrated that nuclear transfer of tadpole nuclei into a
recipient oocyte resulted in the generation of clonally identical
frogs (Gurdon, 1962). In contradiction to the concept classically
illustrated by Conrad Waddington's proposal of the ‘epigenetic
landscape,’ re-depicted here in Fig. 1, this work demonstrated that
the developed characteristics of somatic cells are not fixed
(Ladewig et al., 2013; Waddington, 1957). That is, unlike Weismann's barrier theory, which stated that unwarranted genetic information is erased in cells committed to a certain state
(Weismann, 1893), Gurdon's nuclear transfer experiments showed
that not only do somatic cells maintain all genetic material, but
they can also be reignited by artificial manipulations allowing for
the return to a state of pluripotency. Shortly after Gurdon's
groundbreaking work with nuclear transfer, many other groups
started to present more novel findings pertaining to stem cells. Dr.
James Till and Dr. Ernest McCulloch provided data to prove the
existence of stem cells by injecting healthy marrow cells into irradiated mice and observing the presence of undifferentiated cellular
colonies on the recipients' spleens (Till and McCulloch, 1961). Two
decades after Till and McCulloch's finding, Drs. Martin Evans and
Matthew Kaufman reported their ability to successfully culture
pluripotent cell lines from mouse blastocysts and differentiate
these cells both in vitro and in vivo (Evans and Kaufman, 1981). In
the following decade, Dr. Ian Wilmut made international headlines
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when he used Dr. Gurdon's nuclear transfer method to clone Dolly
the sheep by replacing the nucleus of a fertilized embryo with the
nucleus of an adult mammary gland cell (Campbell et al., 1996). In
1998 Dr. James Thomson further contributed to the stem cell field
by successfully isolating human embryonic stem cells (Thomson
et al., 1998). Together, these findings along with many other reports paved the way for modern stem cell research and regenerative medicine.
1.1. Stem cells
Stem cells are defined as cells that have an unlimited capacity
for self-renewal and are capable of differentiating into multiple cell
types. They are often categorized according to 1) potency, or the
Fig. 1. The epigenetic landscape of somatic cells. Conrad Waddington proposed that
pluripotent stem cells terminally differentiate into somatic cells. It is now known that
pluripotent stem cells undergo stepwise differentiation (blue arrows), producing
lineage-specific progenitor cells that go on to further differentiate into germ layerspecific progenitor cells (EndoSP, MesoSP and EctoSP). These layer-specific progenitor cells then produce cells that comprise germ layer-specific organs of the endoderm
(liver), mesoderm (heart) and ectoderm (brain), to name a few examples. Stem cell
research pioneers, like Gurdon and Yamanaka, demonstrated that somatic cells from
any germ layer could be reprogrammed into pluripotent stem cells (green arrow) and
directly converted from one somatic cell type into another somatic cell type (purple
arrow). PS: pluripotent stem cell, LSP: lineage-specific progenitor, EndoSP: endodermspecific progenitor, MesoSP: mesoderm-specific progenitor, EctoSP: ectoderm-specific
progenitor.
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
3
degree or capacity in which they can differentiate into multiple
cells types or lineages, and 2) origin of isolation.
immune suppression may be required to prevent immunemediated rejection.
1.1.1. Cellular potency
Totipotent stem cells have unlimited capacity and are thus able
to differentiate into either embryonic or extra-embryonic tissues.
Pluripotent stem cells have the capacity to form embryonic tissue
and thus the embryonic cellular lineages: ectoderm, mesoderm,
and endoderm. Multipotent stem cells have the ability to differentiate into a limited number of cell types, which is controlled by a
predetermined level of earlier differentiation.
1.2. Induced pluripotent stem cells (iPSCs)
1.1.2. Origin of isolation
Stem cells may also be defined based on their origin. Human
embryonic stem cells (ESCs) are harvested from the inner cell mass
of the blastocyst of 5-day-old pre-implantation embryos. As ESCs
are pluripotent, i.e. can differentiate into the three germ layers, they
are a popular tool used in regenerative medicine. Fetal stem cells,
unlike ESCs, are isolated later in development, i.e. collected from
fetal tissue such as the developing retina (Aftab et al., 2009;
Baranov et al., 2014), and are multipotent. Adult stem cells
(ASCs), the most developmentally restricted of the 3 cell types, are
suggested to be present in small numbers in most major organs.
These cells can be multipotent, i.e. give rise to a limited number of
cell types specific to the tissue from which they were derived. For
example, ASCs can be found in bone marrow, intestinal crypts and
in the eye near the limbal region posterior to the cornea (Davanger
and Evensen, 1971; Jiang et al., 2002; Snippert et al., 2010).
ESCs have played a substantial role in disease modeling and
treatment studies in the past decade. However, harvesting ESCs
from the inner cell mass of the blastocyst during development restricts their clinical practicality due to limited availability and
ethical concerns. The same can be said for fetal cells, which are
typically isolated even later in embryonic development. In addition,
as ESCs and fetal cells are by definition non-autologous, i.e. not
derived from the patient for which they are destined, for cell
transplantation an additional obstacle of immune incompatibility
exists. For strategies focused on the use of these cell types, life long
Shinya Yamanaka and colleagues revolutionized the stem cell
field in 2006 when they demonstrated that murine fibroblasts
could be reprogrammed into ESC-like pluripotent stem cells,
termed induced pluripotent stem cells (iPSCs) (Yamanaka and
Takahashi, 2006). They used the knowledge that somatic cell
reprogramming can be achieved by transferring a somatic cell's
nucleus into an oocyte in order to investigate if the factors needed
to maintain ESC identity could perform vital functions in the
initiation of pluripotency in somatic cells. Many transcription factors, including Oct3/4, Sox2, and Nanog, work to maintain pluripotency in early embryos (Avilion et al., 2003; Chambers et al., 2003;
Mitsui et al., 2003; Nichols et al., 1998). Other genes that are
commonly up-regulated in tumors, like Stat3, E-Ras, C-myc, Klf4,
and b-catenin, are also known to be involved in the long-term
maintenance of ESC pluripotency and the rapid proliferation of
ESCs in vitro (Cartwright et al., 2005; Kielman et al., 2002; Matsuda
et al., 1999; Phillips et al., 2012). Yamanaka successfully identified
four transcription factors, Klf4, Oct3/4, Sox2, and c-Myc (KOSM),
which were sufficient for successful reprogramming of mouse
embryonic and adult fibroblasts back to a pluripotent state (Fig. 2).
Shortly after the original publication, Yamanaka demonstrated that
this process could be repeated using human fibroblasts, thus
making his work relevant to human disease and an incredibly
promising potential resource for cellular transplantation studies
(Takahashi et al., 2007). Following these original publications, a
plethora of studies focused on the use of different cell types,
reprogramming factors and delivery methods were developed
(Carey et al., 2009; Gonzalez et al., 2009; Jin et al., 2012; Kim et al.,
2009, 2011; Tucker et al., 2013a; Warren et al., 2012; Yoshioka et al.,
2013; Yu et al., 2007; Yulin et al., 2012). Such variations were aimed
at reducing the need for virally induced genetic insertion of the
reprogramming factors, especially the potentially tumorigenic
oncogene, c-Myc.
Fig. 2. Reprogramming somatic cells into iPSCs. The Yamanaka reprogramming factors (Klf4, Oct4, Sox2, c-Myc) are virally transduced into somatic cells, like murine fibroblasts
(pink cells). The reprogramming factors may be packaged in lentiviral vectors, which integrate into the host DNA, or in Sendai viral vectors, which do not require DNA integration.
The transduced cells are reseeded onto feeder cells, such as mouse embryonic fibroblasts (MEFs; blue cells), and fed iPSC induction media containing the pluripotency factor,
leukemia inhibitory factor (LIF). Colony formation (green cells) will occur approximately one week post-transduction, allowing for the individual colonies to be manually isolated
and subsequently expanded into iPSC lines.
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
4
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
The discovery of the iPSC ushered in a new age in the field of
regenerative medicine. Unlike ESCs, the human iPSC is not hindered
by ethical disputes, and, although not yet fully tested, they should
be safer immunologically. That is, a key advantage of the iPSC
technology is that these cells can be generated in large numbers
using cells taken from the patients for which they are intended.
Hence, iPSCs are patient-specific. Additionally, like ESCs, iPSCs are
pluripotent and can be differentiated into any cell type of the three
embryonic germ layers.
The ability to generate patient-specific iPSCs creates novel
avenues for disease modeling and transplantation studies.
Although the iPSC technology holds great promise, a concern of
this technology is that the transgenes used for reprogramming
could remain constitutively active or be randomly incorporated
into the host genome. Retro- and lentiviral platforms commonly
used to deliver reprogramming factors to somatic cells requires
DNA to be integrated into the host's genome. Such genomic
integration can disturb the normal expression and function of
host genes. These concerns have, in large part, been addressed
by use of non-integrating delivery systems such as episomal
viruses (e.g. Sendai virus), which allow reprogramming factors to
be introduced into somatic cells without integrating into the
host genome (Ban et al., 2011; Fusaki et al., 2009; Tucker et al.,
2013a). Unlike MMLV-based retroviruses or HIV-based lentiviruses, Sendai viruses are RNA-based and function independently
of host cell DNA replication. Consequently, transduction with
Sendai virus is transient and viral particles can be removed from
the infected cells following passage and sub-cloning (Ban et al.,
2011; Fusaki et al., 2009). In addition to non-integrating viral
delivery systems, several non-viral reprogramming protocols
have also been developed. The most promising of these are
transient transfection with episomal plasmid DNA (Fontes et al.,
2013; Hu and Slukvin, 2013; Okita et al., 2013) and synthetic selfreplicating RNA (Yoshioka et al., 2013). The latter, developed by
modifying a noninfectious self-replicating Venezuelan equine
encephalitis viral RNA replicon, is a stable DNA-independent
single-stranded RNA molecule that was engineered to carry 4
separate reprogramming factors (Yoshioka et al., 2013).
Following a single transfection of human fibroblasts efficient
generation of integration free iPSCs was achieved (Yoshioka
et al., 2013).
To give a concise overview of the 3 major categories of stem cells
and their advantages and disadvantages from an ophthalmological
standpoint, a summary of the above-described points is presented
in Table 1.
2. Disease modeling using iPSCs
2.1. Mendelian retinal diseases
Being able to offer clinical intervention for inherited genetic
disorders is greatly dependent on the understanding of a patient's
disease-causing mutations and the consequential pathophysiological mechanism(s) that these disease-causing mutations induce.
Lacking the knowledge of a patient's disease-causing gene and how
the genetic mutations impact the health and behavior of the
affected cells, it would be nearly impossible to generate and provide patient-specific therapeutic strategies. Apart from being a
promising cell source for autologous transplantation, a major
advantage of the iPSC technology is that it provides the ability to
model and study human disease in vitro. In particular, iPSCs are a
useful tool for 1) discovery and molecular confirmation of newlyidentified disease-causing mutations; 2) the interrogation of disease pathophysiology in relatively inaccessible tissues such as the
retina that cannot be routinely subjected to molecular analysis in
living patients; and 3) the rapid testing of novel disease and
patient-specific therapeutics, especially important for rare diseases
in which animal models that accurately recapitulate the disease
phenotype do not exist.
As depicted in Fig. 3, the ability to develop a better understanding of disease mechanism will increase the number of therapeutic avenues available to clinicians. The following sections
provide some examples of the ways in which iPSCs have been used
to model Mendelian eye diseases such as MAK-associated retinitis
Table 1
Major stem cell categories and their advantages and disadvantages for development of ocular therapy.
Category
Embryonic/fetal
Adult
Induced
Derivation/generation
Derived from developing embryos
(e.g. blastocyst or developing fetus)
Derived from developmentally mature organs
Generated from terminally differentiated tissue
Examples
Induced pluripotent stem cell
Advantages
Able to generate retinal cells,
including photoreceptors, RPE,
and choroidal endothelial cells
Have unlimited self-renewal,
so single donations offer enough
cells for large number of transplantations
No ethical concerns
Potential to be patient-specific, less concern
for immune rejection
Do not form tumors
Capable of generating autologous
patient-specific retinal cells including
photoreceptors, RPE, and choroidal
endothelial cells
Unlimited capacity for self renewal
Less concern of immune rejection
No ethical concerns.
Ability to recapitulate disease in a dish for
study of pathophysiology and testing of
therapeutic efficacy
Disadvantages
Ethical concerns regarding source
Not patient-specific
Have the potential to form tumors if not
properly differentiated and isolated
Limited potency
Limited capacity for self-renewal
Have the potential to form tumors if not
properly differentiated and isolated
Depending on disease phenotype,
genetic correction of patient specific
mutations may be required
Embryonic stem cells
Retinal progenitors
Subventricular zone stem cells
Photoreceptor precursor cells
Mesenchymal stem cell
Hematopoietic stem cell
Intestinal crypt stem cell
Skin derived precursor cell
Limbal stem cells
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
5
2.2. MAK-associated retinitis pigmentosa
Fig. 3. Schematic depicting how therapeutic modalities available depend on knowledge and progression of retinal degenerative disease in question. A major goal of using
patient-specific iPSCs for disease modeling is to acquire more knowledge of the disease's pathogenesis to effectively prevent the retinal degeneration from progressing.
The greater the amount of knowledge known about a particular retinal degeneration
allows for the development of preventative therapies. For instance, if a disease-causing
mutation is known, pre-implantation testing prior to in vitro fertilization could be
performed to implant ESCs without the disease-causing mutation. However, corrective
treatments are needed as the amount of disease knowledge decreases (green arrow)
and disease progression occurs (purple arrow). These avenues start with drug therapies that may slow disease progression. If the genetic cause of a disease is known, gene
and/or cell replacement therapy may be a viable option. For late-stage disease, more
invasive therapies like optogenetics or retinal prosthesis may be necessary.
pigmentosa, Usher Syndrome Type II, CEP290-associated Leber
congenital amaurosis, juvenile neuronal ceroid lipofuscinosis, and
Best disease, as well as the complex heterogenous disorder, agerelated macular degeneration.
Although some inherited retinal diseases are well-understood
mechanistically, like ABCA4-associated Stargardt disease, there are
still a large number of patients whose disease-causing genes are
either unknown or who have known mutations in genes with
poorly understood functions. Retinitis pigmentosa (RP) is a particularly devastating type of blinding disease because it gradually
claims a person's vision over many years and in some cases also
threatens the vision of family members. One of the many obstacles
to making gene- and cell-based therapies an everyday reality for
people afflicted with RP is the extensive genetic heterogeneity of
the disease. To date there are over 60 different genes that have been
associated with RP, which can occur in the retina alone or together
with other syndromic disorders (Petrs-Silva and Linden, 2014). In
our experience, these 60 genes account for less than half of all cases
of the disease. That is, over 163 different genes that cause an RP-like
phenotype have been identified via genetic sequencing of patients
at the University of Iowa Carver Non-profit Genetic Testing Lab
(unpublished data). Thus, it is not uncommon for a newly discovered RP gene to cause less than 1% of all cases of the disorder. To
make a major impact in the RP field, scientists will need to develop
ways to rapidly translate a success achieved in treating one genetic
subtype of RP into the treatment of another. That is, they will need a
series of interchangeable and reusable reagents and strategies.
Recently we used patient-specific iPSC-derived photoreceptor
precursor cells generated from patients with retinal degenerative
diseases to show that a homozygous Alu insertion, identified in
exon 9 of the gene male germ cell-associated kinase (MAK), results
in loss of the transcript and an inability to produce functional MAK
protein (Tucker et al., 2011b). Loss of MAK was found to disrupt
normal photoreceptor cell structure leading to cell death and
irreversible blindness. In addition to allowing us to confirm pathogenicity of the MAK mutation, we were also able to identify and
confirm the involvement of a unique MAK-specific retinal transcript
in disease pathophysiology. This transcript contains an extra 75
base pair exon (exon 12), which is phylogenetically conserved and
is only expressed in transcripts that also contain MAK exon 9
(Tucker et al., 2011b). Although mutations in MAK are an uncommon cause of RP in the general population (about 1%) they are quite
common among individuals of Ashkenazi Jewish ancestry, accounting for as much as one third of this disease in the Jewish
population (Stone et al., 2011). Collectively these findings have
enabled progress in the development of gene and autologous cellbased interventions for MAK-associated RP in which patientspecific iPSC-derived photoreceptor cells can be used to confirm
therapeutic efficacy in vitro and replace lost cells in vivo. As an
additional example of iPSC-derived retinal cells being used to test
gene-based therapeutics, a recent study published by Tsang and
colleagues show efficacy of AAV8-mediated delivery of Membrane
Frizzle-related Protein (MFRP) to rescue the cellular phenotype
observed in RPE cells with MFRP-associated RP (Li et al., 2014).
2.3. Usher syndrome type II
In a similar study, iPSC-derived photoreceptor precursor cells
were used to confirm two disease-causing mutations, one being a
novel intronic mutation in the gene USH2A in an adult patient with
autosomal recessive RP (Tucker et al., 2013b). In this study,
keratinocyte-derived iPSCs were differentiated into multi-layer
eyecup-like structures that contained an outer layer of RPE and
an inner layer of photoreceptor-like neuronal cells. These photoreceptor precursor cells expressed photoreceptor-specific markers,
recoverin and rhodopsin and exhibited axonemes and basal bodies
structurally characteristic of photoreceptor outer segments (Tucker
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
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L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
et al., 2013b). Further analysis of the identified USH2A transcripts in
these cells revealed that one of the patient's mutations causes
exonification of intron 40, a translation frameshift and a premature
stop codon. Increased expression of the endoplasmic reticulum
(ER) stress-related proteins, GRP78 and GRP94 suggested that the
patient's other USH2A variant (a single point mutation resulting in
an Arginine to Histidine transition at position 4192) causes disease
through protein misfolding and ER stress. To further support the
idea that this disease is ER stress-related rather than a developmental defect, photoreceptor precursor cells were transplanted
into 4-day-old immunodeficient Crb1/ mice. Transplanted
patient-derived cells integrated and formed morphologically and
immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient cause degeneration of
developmentally normal photoreceptors over time. The role of ER
stress in RP-associated photoreceptor cell death is consistent with
previous findings in other disease causing mutations. For example,
in a similar study Jin and colleagues were able to show that iPSCderived rod photoreceptor precursor cells generated from a patient with autosomal dominant RHODOPSIN-associated RP have
elevated levels of ER stress-related proteins and subsequently
succumb to apoptotic cell death in vitro (Jin et al., 2011). Unlike
MAK-associated RP, neither USH2A nor RHODOPSIN-associated
diseases will be amenable to the traditional gene augmentationbased approach (Burnight et al., 2014; MacLaren et al., 2014;
Maguire et al., 2009, 2008; Vasireddy et al., 2013). That is, with a
coding region of ~19,000 bp, full-length USH2A exceeds the packaging capacity of either of the two viral delivery systems used in the
eye to date (i.e. AAV packaging limit is ~4000 bp, lentiviral packaging limit is ~10,000 bp). For this disease, delivery systems with
larger carrying capacity could be effective. That said, cloning of
mutation-free genes of this size is often problematic. For autosomal
dominant diseases such as RHODOPSIN-associated RP, in which the
disease-causing mutation causes a gain-of-function, deletion or
knockdown of the mutated allele will likely be required. As discussed in detail below, a promising new approach that may be
useful for treating both of these classes of disease is CRISPR-based
genome editing.
2.4. CEP290-associated Leber congenital amaurosis
Leber congenital amaurosis (LCA) refers to a collection of
inherited retinal degenerative disorders characterized by
nystagmus, little to no recordable electroretinogram, and poor
vision presenting from birth (Coppieters et al., 2010). LCA is predominantly inherited in an autosomal recessive manner and of the
seventeen loci associated with the disease, the largest contributor
is CEP290 (Perrault et al., 2007; Stone, 2007). Thirty percent of LCA
patients carry mutations in CEP290, the gene product of which is a
cilia-associated protein that functions in photoreceptor outer
segment trafficking and ciliogenesis (Craige et al., 2010; Drivas
et al., 2013; McEwen et al., 2007; Tsang et al., 2008). Loss of
photoreceptor outer segments in patients with CEP290-associated
LCA results in severe vision loss. However, some patients retain
cone photoreceptors in the central fovea and MRI studies indicated
that visual pathways of the inner retina in these patients display
normal architecture (Cideciyan et al., 2007). These data, as well as
the recent successes garnered in gene augmentation therapy trials
for RPE65-associated LCA, support gene and cell therapy as an option to treat CEP290-associated LCA. Patient-specific iPSC-derived
photoreceptors provide an excellent opportunity to study therapeutic strategies in vitro.
Recently, we demonstrated a ciliogenesis defect in CEP290associated LCA patient cells (Burnight et al., 2014). Previous work
indicated that the rd16 mouse model of CEP290-associated LCA
exhibited defects in number and length of cilia in serum-starved
fibroblasts (Drivas et al., 2013). When we investigated ciliogenesis
in CEP290-associated LCA patient fibroblasts with various CEP290
mutations, we observed a decrease in the number of cells forming
cilia and in the length of cilia formed. This cilia phenotype was not
observed in cells from every CEP290 patient, presumably due to the
location and severity of the mutation within the gene. In addition,
following gene augmentation, we were able to show that the range
of therapeutic dosage was very narrow, i.e. overexpression of wildtype CEP290 itself was toxic. The ability to determine pathophysiology and mutation-specific disease severity in individual patients
will give us the opportunity to tailor treatments thereby providing
more efficacious outcomes.
2.5. Juvenile neuronal ceroid lipofuscinosis
In addition to gene and new gene mutation discovery, iPSCs can
be utilized to interrogate disease mechanism, discover potential
drug therapies and to evaluate the efficacy of gene correction approaches in human cells. We are currently using iPSCs to gain
insight into the devastating autosomal recessive inherited disease,
juvenile neuronal ceroid lipofuscinosis, more commonly known as
Batten disease. Batten disease is a rare and fatal lysosomal storage
disorder that is most often caused by a one-kilobase genomic DNA
deletion in the gene, ceroid lipofuscinosis 3 (CLN3) (de los Reyes
et al., 2004). Ophthalmologists typically diagnose Batten disease
as it presents with severe deficits in central visual acuity due to loss
of cone photoreceptors in the macula. Batten disease is usually
diagnosed early in a child's life, between the ages of five to seven.
Although visual defects and blindness are the first to arise, patients
eventually experience seizures, difficulty communicating and
walking and severe mental deficit due to extensive central nervous
system neuronal cell death. Batten patients face an extremely
difficult life of blindness, epilepsy, are often bedridden and usually
die during the second or third decade of life. There are currently no
treatment options available and little is understood about the
molecular role of CLN3 or the mechanism of disease onset when
CLN3 is mutated (Seehafer et al., 2011).
A key feature of Batten disease is the cytoplasmic accumulation
of lysosomal autofluorescent material within neurons (lipofuscin)
(Gardiner et al., 1990). We are taking advantage of this easily
identifiable phenotypic disease characteristic to discover drug
treatment options for Batten patients. We have generated patientderived iPSCs and retinal precursor cells from skin biopsies of human patients with molecularly confirmed CLN3-associated Batten
disease. Into these patient-derived cells we are also introducing an
apoptosis-dependent reporter construct (Bardet et al., 2008). We
are using these diseased patient reporter cells to perform highthroughput drug screens to look for compounds that are capable
of mitigating the accumulation of lysosomal lipofuscin deposits and
that prevent neuronal apoptosis, thus protecting patient-derived
photoreceptor precursor cells from CLN3-associated cell death.
Promising compounds can then be further characterized in vivo for
their ability to ameliorate lysosomal storage deposits and retinal
degeneration in Cln3 knockout mice (Katz et al., 1999; Mitchison
et al., 1999). Utilizing autologous stem cells for approaches
similar to this one could be a good avenue in which to discover new
treatment modalities in addition to providing novel insights into
disease mechanisms for previously untreatable diseases.
2.6. Best disease
Unlike RP, which is usually caused by mutations in genes
expressed in photoreceptor cells, Best disease results from mutations in the RPE-specific gene, BEST1 (Petrukhin et al., 1998).
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
Best disease selectively affects the region of the human retina
within one disk diameter of the cone photoreceptor-rich fovea
and predisposes affected individuals to the development of vision
threatening choroidal neovascular membranes secondary to
vitelliform lesions (Kay et al., 2012). Late stages of Best disease are
often marked by geographic loss of the RPE underlying the macula
and gliotic scarring in the neural retina (Mullins et al., 2007,
2005). Although mutations in BEST1 have long been known to
cause Best disease, as mouse models of disease lack the major
disease phenotypes, the mechanism by which these mutations
cause onset of disease remained unclear. In an elegant study
recently published by Singh et al., human iPSC-derived RPE were
generated from patients with Best disease and unaffected siblings
and used to examine the cellular and molecular disease processes
(Singh et al., 2013). They observed that RPE derived from patients
with Best disease displayed disrupted fluid flux and increased
accumulation of autofluorescent material after long-term feeding
of photoreceptor outer segments (Singh et al., 2013). Furthermore,
chronic photoreceptor outer segment feeding led to delayed
rhodopsin degradation and turnover, cellular responses to calcium
were sensitized, and oxidative stress levels were altered
(Abramoff et al., 2013; Singh et al., 2013), cell biological findings
consistent with altered outer segment-RPE interactions observed
in living Best disease patients (Abramoff et al., 2013). Collectively,
these findings suggest that BEST1 plays an important role in
photoreceptor outer segment turnover and homeostasis and implicates disruption of this pathway in the pathogenesis of Best
disease. These studies point to a variety of therapeutic avenues, in
particular drug-based studies focused on manipulation of
phagocytic and protein degradation pathways may be particularly
promising.
2.7. Using iPSCs to model AMD
Age-related macular degeneration (AMD) is a genetically heterogeneous and complex disease with over 50 different genetic risk
loci identified to date (Gorin, 2012; Liu et al., 2012), 19 of which
meet genome wide significance (Fritsche et al., 2013). Early AMD
typically presents in the sixth decade of life or later with the formation of lipid-like deposits, called drusen, that accumulate
beneath the RPE, as well as drusen-like deposits that appear in the
subretinal space (Curcio et al., 2013). A subset of patients with early
AMD later develops geographic atrophy of the RPE and underlying
choriocapillaris, resulting in the loss of the overlying photoreceptors. Other patients with early AMD develop a complication
sometimes referred to as “wet AMD” in which the underlying
choroidal vasculature grows into the sub-RPE and/or sub-retinal
space forming fenestrated, leaky vascular networks known as
choroidal neovascular membranes (CNVs) (Jager et al., 2008). For
AMD, all of the therapeutic interventions shown in Fig. 3 could
eventually come into play. Drug therapy using the anti-vascular
endothelial growth factor (VEGF) agents Avastin and Lucentis
have proven very efficacious in ameliorating CNVs, promoting fluid
resorption, and slowing vision loss in wet AMD (Agosta et al., 2012;
Scott and Bressler, 2013; Stone, 2006). As AMD is genetically
heterogenous, traditional gene augmentation approaches seem less
promising than for Mendelian diseases. However, cell replacement
therapy for patients that have lost photoreceptors, RPE and
choroidal (choriocapillaris) endothelial cells is being pursued by a
number of groups and, if successful, would be extremely beneficial
for thousands of patients worldwide (Kamao et al., 2014; Kokkinaki
et al., 2011; Liao et al., 2010; Zhu et al., 2011). In very late stage AMD,
in which significant cell loss has occurred, optogenetic and retinal
prosthetic approaches may eventually be viable therapeutic options, although the resolution of the latter devices will need to be
7
improved if they are to offer AMD patients a significant improvement in visual acuity.
The greatest opportunity for AMD treatment lies in understanding the genetic causes and pathogenic mechanisms thoroughly enough to be able to identify those with a greater
propenstity to develop the disease and prevent it from happening
through early and efficacious prophylactic intervention. Although
the notion that AMD has a strong genetic component was advocated as early as the 1970s (Davanger and Evensen, 1971; Gass,
1973; Jiang et al., 2002; Snippert et al., 2010), our understanding
of the biological mechanisms through which specific genetic loci
contribute to the development and progression of AMD is still
poorly understood. Genome wide association studies of AMD
identified two loci on chromosomes 1 and 10 that are highlyassociated with disease risk (Edwards et al., 2005; Haines et al.,
2005; Jakobsdottir et al., 2005; Klein et al., 2005; Rivera et al.,
2005; Schwartz et al., 2012b; Yamanaka and Takahashi, 2006;
Zareparsi et al., 2005). The risk associated with the chromosome
1q locus is predominantly due to a haplotype that harbors a
missense mutation (Tyr402His) in the complement factor H (CFH)
gene (Avilion et al., 2003; Chambers et al., 2003; Hageman et al.,
2006; Mitsui et al., 2003; Nichols et al., 1998; Zhang et al., 2008).
The complement cascade was implicated in the pathogenesis of
AMD prior to the association of CFH variants with the disease (Bora
et al., 2005; Cartwright et al., 2005; Johnson et al., 2001, 2000;
Kielman et al., 2002; Matsuda et al., 1999; Mullins et al., 2001,
2000; Phillips et al., 2012), and thus the involvement of this inhibitor in AMD fits into a mechanistic framework of impaired
complement regulation and bystander cell injury (Mullins et al.,
2011a; Tucker et al., 2011b). In contrast, the mechanism by which
the chromosome 10q locus increases AMD risk remains unknown
and controversial. The most common high-risk haplotype contains
two genes (ARMS2 and HTRA1) with plausible disease-causing
mutations. The variants include a non-conservative polymorphism in the ARMS2 gene (Ala69Ser), a complex 144 bp deletion and a 54 bp insertion in the 30 untranslated region of the
ARMS2 transcript, and a promoter polymorphism in the HTRA1 gene
(Fritsche et al., 2008; Stone et al., 2011).
These variants are in strong linkage disequilibrium such that
only a few percent of 10q alleles harboring ARMS2 Ala69Ser lack the
HTRA1 promoter variant and vice versa. This, coupled with the
overall genetic complexity of AMD and a lack of representative
animal models, have made it difficult to determine which, if either,
of these two genes is responsible for conferring the AMD risk
associated with the 10q locus. Recently, Yang and colleagues
employed an unbiased proteome screen of N-retinylidene-N-retinyl-ethanolamine (A2E)-aged patient-specific iPSC-derived RPE
cell lines derived from patients with high and low risk 10q26 loci
(Coppieters et al., 2010; Yang et al., 2014). They reported that cells
from patients who were homozygous for the high-risk haplotype
had a reduction in superoxide dismutase 2 (SOD2)-mediated antioxidative defense. The authors concluded that the ARMS2/HTRA1
risk alleles decrease SOD2 defense, leaving RPE more susceptible to
oxidative damage and thereby contributing to AMD pathogenesis.
By elucidating the disease mechanism(s) associated with this locus,
new targets for therapeutic intervention have potentially been
identified.
Historically, the RPE has been seen as the key cell type involved
in AMD pathogenesis. Currently, clinical trials focused on stem cellderived RPE cell transplantation are underway. The first, initiated
by Robert Lanza and colleagues at Advanced Cell Technology in the
UK, was designed to treat patients with dry AMD and Stargardt
disease. In this trial patients receive bolus subretinal injections of
human embryonic stem cell-derived RPE (NCT01344993). Initial
short term reports indicate that transplanted cells attached and
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
8
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
persist within the subretinal space (Schwartz et al., 2012a). Pete
Coffey, Dennis Klegg and colleagues have devised a similar trial
focused on subretinal transplantation of human ESC-derived RPE
cell sheets for the treatment of patients with AMD (NCT01691261).
Similarly, in Japan, Masayo Takahashi and collaborators at the
RIKEN Center for Developmental Biology have recently transplanted autologous iPSC-derived RPE cell sheets into a woman with
AMD (JPRN-UMIN000011929). The latter two studies have focused
on sheet transplants rather than bolus cell injection, which have
been shown to dramatically improve survivability and integrative
capacity (Hu et al., 2012). Cell suspension injections may lead to the
formation of isolated cellular clumps that fail to develop into
polarized RPE monolayer (Lu et al., 2001; Phillips et al., 2003;
Shiragami et al., 1998).
To date, the contribution of the choroid, in particular the underlying choriocapillaris vasculature that supports the RPE and
photoreceptor cells, has not been thoroughly studied and remains
unknown. This lack of knowledge is largely due to the historical
inability to clinically image this tissue for better understanding its
role in pathogenesis. Recent advances in imaging technology now
allow for live imaging, measurement and assessment of choroidal
thickness using spectral-domain optical coherence tomography
(SD-OCT). Previous studies using the SD-OCT technique on eyes
with early-stage AMD without CNVs showed a thinning of the
choroid when compared to healthy age-matched controls (Hu et al.,
2013; Ko et al., 2013; Sohn et al., 2014).
Although it is undeniable that the RPE plays an important role in
AMD pathogenesis, findings from our group and others have shown
that there is loss of choriocapillaris endothelial cells in eyes with
both early- and late-stage AMD (Grunwald et al., 2005; McLeod
et al., 2009; Ramrattan et al., 1994). For instance, Sohn and colleagues recently reported that human donor eyes known to have
dry AMD are associated with choroidal thinning and that AMD
choroids have higher levels of tissue inhibitor of metalloproteinase
3 (TIMP3), which is associated with Sorby's fundus dystrophy, an
autosomal-dominant form of macular degeneration (Sohn et al.,
2014; Visse and Nagase, 2003). Additionally, a gene set enrichment analysis (GSEA) study on RPE-specific and endothelial cellassociated gene sets found that RPE transcripts were preserved
and increased in early AMD, along with a significant decrease of
endothelial cell marker expression, suggesting that choroidal
endothelial cell dropout is an early event in the pathogenesis of
AMD (Whitmore et al., 2013), consistent with morphometric
studies showing increased numbers of choriocapillaris ghost vessels with increasing abundance of drusen (Fig. 4A) (Mullins et al.,
2011b). These findings indicate that choroidal endothelial cell
preservation may slow the progression of or help protect against
the onset of early AMD.
Stem cell-derived choroidal endothelial cells for the purpose of
cellular transplantation may also be required for the reconstruction
of the macula in advanced AMD. Although the loss of choriocapillaris EC in early AMD is notable, in endstage AMD (geographic
atrophy or choroidal neovascularization) choriocapillaris dropout is
profound (Fig. 4B). In advanced AMD, a combined approach in
which photoreceptor cells, RPE and choriocapillaris EC are replaced
may be necessary in order to restore vision.
Endothelial cells can be generated from iPSCs through coculture with primary endothelial cell lines or conditioned media
with endothelial cell-specific factors (Choi et al., 2009; Du et al.,
2014). For instance, Park et al. successfully generated cord blood
iPSC-derived vascular progenitors and used these cells to repair
damaged retinal blood vessels (Park et al., 2014b). However, to our
knowledge there is no published data showing the differentiation
of iPSC-derived choroidal endothelial cells. The choroid is structurally distinct from other vascular tissues, like the brain
Fig. 4. Choriocapillaris dropout and membrane attack complex in eyes with macular
degeneration. (A) Immunofluorescent labeling of the complement membrane attack
complex (green) in a human eye with early/dry AMD. Note the robust labeling at the
level of the choriocapillaris (CC) and relative sparing of the RPE. Sections were dual
labeled with UEA-I, a marker of viable endothelial cells (red) in the choroid of an eye
with dry AMD. Note the abundant “ghost” vessels in the choriocapillaris (asterisks),
that are unoccupied by viable endothelial cells. Choriocapillaris loss in eyes with
advanced AMD is striking, as depicted in an eye with choroidal neovascularization (B).
A comprehensive cell replacement strategy to treat AMD will most likely require
addressing the vascular loss in this disease. BlamD: layer of basal laminar deposit, RPE:
retinal pigment epithelium, CC: choriocapillaris, CH: outer choroid; RET: neural retina.
microvasculature or aortic vasculature, due to its broad, fenestrated
and flat lumens with diameters ranging from 20 to 50 mm (Lutty
et al., 2010). Nevertheless, the extent to which choroidal endothelial cells are molecularly unique compared to other endothelial cells
is not well understood. It is possible that the microenvironment of
the choroid causes residing hemangioblasts, precursors of hematopoietic and endothelial cells, to develop and maintain the
unique choroidal endothelial cell features. For example, it is
thought that VEGF secreted from the overlying RPE is necessary in
maintaining the fenestrations seen in the choriocapillaris
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
(Blaauwgeers et al., 1999; Lutty et al., 2010). In order to develop
successful treatments for macular degenerations involving
choroidal disruption, it is critical to understand if the choroidal
endothelial cells vary from other endothelial cells. Therefore, future
studies are needed to characterize choroidal endothelial cells to
determine if they are unique compared to other endothelial cells at
the molecular level.
Although the replacement of choroidal ECs derived from iPSCs
poses many challenges, there is basis to suspect that autologous EC
may be well tolerated in the choroid. IPSC-derived ECs have been
shown to integrate with the host vasculature and trigger a relatively modest immune response in a murine model of hindlimb
ischemia (de Almeida et al., 2014). Transplanted iPSC-derived ECs
may similarly recanalize ghost vessels in the choriocapillaris. This is
an active area of investigation.
As mentioned above, complement activation has long been
shown to play a major role in the pathogenesis of AMD. This notion
is further substantiated in that the complement cascade is also
active in the choriocapillaris of high-risk CFH AMD eyes (Mullins
et al., 2011a). It is possible that complement complexes near the
choriocapillaris put stress on the choroidal endothelium and help
cause choriocapillaris loss reported in early AMD (Mullins et al.,
2011a). The majority of complement membrane attack complexes
(MAC) are present in domains surrounding the choriocapillaris e
not the RPE or photoreceptors (Fig. 4A) (Mullins et al., 2014). These
results further demonstrate the need to elucidate the choroid's role
in AMD pathogenesis that could contribute to the development of
new treatments for AMD. Development and implementation of
efficient choroidal endothelial cell differentiation protocols will
empower further studies focused on modeling aspects of AMD
specific to the choroid.
Stem cell-derived therapeutic modalities are also showing
promise for the treatment of another blinding eye disease, glaucoma. The laboratory of Donald Zack has used iPSC-derived ganglion cells to perform high-throughput drug screens for agents that
prevent ganglion cell death in glaucomatous cells and for assessing
potential neuroprotective growth factors secreted by iPSC-derived
retinal ganglion cells (Sluch and Zack, 2014). Also, John Fingert
and collaborators recently demonstrated the generation of patientspecific iPSC-derived ganglion cells that have a duplication of the
gene, TBK1, which causes normal tension glaucoma. In this study
patient-specific iPSC-derived retinal cells were used to show that
TBK1 duplication causes increased activation of the autophagy
pathway (Tucker et al., 2014).
In the following sections, we will review drug, gene replacement, genome editing and cell replacement strategies being
employed by our group and others to treat inherited orphan retinal
diseases. Included, will be a discussion regarding the debate of eye
immune privilege and how donor and autologous stem cell transplantation approaches could affect the recipient eye and immunological homeostasis.
3. Gene augmentation and cell replacement treatment
strategies
The heritability of Mendelian retinal degenerative diseases,
while one of their most frightening features, is also the means by
which they will be cured. That is, the fact that these diseases are
caused by detectable variations in a gene that is expressed in the
retina will make it possible to 1) lessen the risk of recurrence of the
diseases in affected families through a combination of genetic
testing and genetic counseling, 2) prevent vision loss by early
detection of the disease coupled with gene replacement therapy or
some other form of treatment (e.g., immune modulation or drug
therapy) and 3) create genetically-corrected and immunologically-
9
matched photoreceptor precursor cells that can be transplanted
into the retina to restore vision.
For numerous diseases, especially those involving significant
neurodegeneration or other types of organ failure, drug intervention or gene therapy alone will not suffice. In these conditions,
cellular replacement will be required to rescue, refurbish and
preserve tissue function. Recessive diseases, particularly those that
result in a null phenotype, such as MAK-associated RP and Batten
disease, to name two, are often amenable to gene therapy. That is,
replacement of the defective mutated gene with the normal transcript prior to photoreceptor cell death could potentially arrest
disease progression and prevent subsequent loss of vision. A variety
of gene delivery approaches have been developed and used in the
eye. These range from non-viral, plasmid transfection approaches,
such as cell-penetrating nanoparticles (Read et al., 2010a,b), to viral
vector transduction technologies, such as adeno-associated viruses
€ m et al., 2003; Yang
(AAV) (Maguire et al., 2009, 2008; Narfstro
et al., 2002) and lentiviruses (Auricchio et al., 2001; Pang et al.,
2006).
As genetic testing for rare recessive diseases becomes more
widespread, patients will be definitively diagnosed earlier in the
course of their disease when viral gene replacement therapy has
the greatest opportunity to be efficacious, thus sparing the patient's
vision. However, for patients with more advanced disease, retinal
cell replacement therapy will be necessary. Patient-derived iPSCs
have now made this avenue a realistic option. As we have discussed
above, patient-derived stem cells harbor disease-causing mutations
(Tucker et al., 2013b, 2011b), which make them ideal for disease
modeling but a bit less convenient for cell-replacement therapy.
That is, for many disorders, the patient's disease-causing mutations
must first be corrected prior to transplantation if they are going to
be able to have any lasting positive effects. This relative disadvantage is balanced by the tremendous advantage that patient-derived
iPSCs are immunologically identical to the patient and thus have
the greatest chance of evading the recipient's host immune system
with the smallest amount of immunomodulatory medication.
3.1. iPSCs and gene augmentation
Gene augmentation is an effective strategy to treat inherited
retinal dystrophies, the causes of which are mutations in enzymes
or other small genes easily packaged into the well-characterized
and efficient adeno-associated virus (AAV) vectors. Numerous
clinical trials involving AAV-mediated RPE65 augmentation have
restored vision to patients suffering from RPE65-associated LCA e
an inherited retinal dystrophy resulting from deficiency of the
retinal isomerase, RPE65 (Bainbridge et al., 2008; Hauswirth et al.,
2008; Maguire et al., 2009, 2008). Most recently six patients with
the X-linked recessive blinding disease choroideremia were given
AAV vectors expressing the CHM gene that encodes the Rab escort
protein 1 (REP1). Six-months post-injection, increases in visual
acuity and retinal sensitivity (measured via dark-adapted microperimetry) were consistent with improved rod and cone function in
AAV-treated eyes (MacLaren et al., 2014).
We recently demonstrated successful gene transfer to CEP290associated LCA patient iPSC-derived photoreceptor precursors using lentiviral-mediated gene delivery (Burnight et al., 2014).
Because CEP290 cDNA (~8 kb) is too large to package in AAV vectors
commonly used for clinical gene therapy treatments, we packaged
the full-length CEP290 in a lentivirus which can accommodate
larger transgenes (8e10 kb packaging capacity) (Balaggan and Ali,
2012). Unfortunately but importantly, we found that overexpression of this large structural protein caused increased cell
death in a dose-dependent manner of CEP290-expressing lentiviral
vectors (Burnight et al., 2014). Nonetheless, when using a lower
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
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L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
dose, we were able to rescue ciliogenesis in diseased patient cells
indicating that restoring full-length CEP290 expression may be an
effective treatment option for vision loss in these patients. Due to
the adverse cellular toxicity associated with CEP290 overexpression, future experiments in gene transfer will need to
address this obstacle. Using cell type-specific promoters whose
transcriptional activity is less than that of the strong, viral cytomegalovirus promoter used in our study might decrease the
toxicity resulting from CEP290 gene transfer (Burnight et al., 2014).
Additionally, one could take advantage of the native CEP290 regulatory elements through genome editing strategies to correct the
patient-specific mutation, thereby avoiding ectopic CEP290
expression altogether.
3.2. iPSCs and genome editing
Recent advances in genome editing approaches, including the
use of zinc finger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs), are especially exciting for the potential boost these technologies could provide to cell replacement
therapy (Christian et al., 2010; Hockemeyer et al., 2011; Miller et al.,
2007; Porteus and Baltimore, 2003). Components of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 adaptive immune system can also be used to efficiently
and specifically edit human genes using pre-designed 20 nucleotide small guide RNAs (sgRNA) coupled with a human codonoptimized Cas9 nuclease (Cong et al., 2013; Maeder et al., 2013;
Ran et al., 2013a,b). Similarly to ZFN- and TALEN-mediated gene
correction, cells repair the majority of CRISPR/Cas9-induced double
strand breaks (DSBs) by one of two mechanisms. In the absence of a
homologous repair template, the cell naturally employs nonhomologous end joining (NHEJ) leading to the insertion or deletion (InDel) of sequence surrounding the DSB (Sun et al., 2012).
Repair via NHEJ represents a feasible strategy for intronic mutations in areas of poorly conserved sequence. However, to correct
loci at which large deletions or insertions occur or where NHEJmediated InDels would not be tolerated (i.e. splice sites), the
sgRNAs and Cas9 must be co-delivered with an engineered repair
template containing unmutated, wild-type sequence. DSB repair
occurs via homologous recombination using the exogenous repair
sequence as template. Homology-directed repair (HDR) in diseased
patient cells harboring mutations would presumably restore wildtype gene and protein expression and function. Thus, following
CRISPR-based correction, stem cell-derived retinal cells to be used
for human transplantation would no longer harbor disease-causing
mutations. This technology has the potential to elevate stem cell
replacement therapy from being a stopgap, temporary solution, to
being a long-term disease remedy.
As a proof of principle for the utility of combining genome engineering with the iPSC technology, researchers in the Jaenisch lab
combined engineered ZFNs with iPSC technology to create disease
and control human iPSC lines to study Parkinson disease (Soldner
et al., 2011). Soldner and colleagues designed ZFNs targeting the
a-synuclein locus (SNCA), a gene commonly mutated in Parkinson's
disease (PD). Using four different targeting strategies, the investigators engineered an array of isogenic and control cell lines
that either created two common PD-related mutations (A53T or
E46K) in wild-type ESCs or corrected the mutation in PD patientderived iPSCs.
Soldner et al. initially employed a drug selection-based strategy
to introduce the A53T mutation (a single base pair substitutiondG209Adin exon three of SCNA) into ESCs. The donor
construct carried ~600 bp homology on either side of the wild-type
sequence at nucleotide 209 (G209) and a floxed puromycinresistance cassette just downstream of the mutation in intron
three. Co-electroporation of the ZFN expression plasmids and
donor construct into two ESC lines resulted in 3 out of 336
puromycin-resistant clones targeted to the correct locus. Following
Cre-mediated excision of the selection cassette and sequence
analysis, two of the three clones contained a small deletion in the
second allele as a result of the modification. However, the
remaining correctly targeted clone maintained pluripotency and
was able to differentiate into tyrosine hydroxylase-expressing
neurons (Soldner et al., 2011). The second strategy utilized both
positive and negative selection to introduce the A53T mutation into
both alleles of one of the ESC lines. By incorporating the herpes
simplex virus thymidine kinase (HSV-TK) and diphtheria toxin A
(DT-A) into the donor cassette, 9 of 41 puromycin- and ganciclovirresistant colonies resulted in correct targeting. One of the clones
confirmed by Southern DNA and sequencing analysis contained
modification of the second allele.
A third strategy generated A53T mutant ESCs without drug
selection. The investigators co-delivered ZFNs and a donor
plasmid carrying about 1 kb homologous sequence flanking the
ZFN cleavage site and the A53T (G209A) point mutation. Additionally, Soldner et al. delivered a reporter cassette expressing
eGFP to enrich transfected cells via fluorescence-activated cell
sorting (FACS). Analysis of single-cell-derived clones via Southern
blotting and PCR-mediated genotyping revealed one of 240
clones was correctly targeted. Cells from the clone maintained
pluripotency and were able to differentiate into dopaminergic
neurons (Soldner et al., 2011). Instead of using a double-stranded
plasmid donor vector, the fourth strategy employed a short
single-stranded oligodeoxynuceotide (ssODN) donor construct as
a repair template to introduce the PD-associated E46K (G188A)
point mutation into ESCs. Using the aforementioned FACS
enrichment strategy, the researchers successfully recovered five
of 720 single-cell-derived clones with the correct insertion of the
E46K/G188A mutation.
Lastly, the investigators employed ZFN-mediated genome editing to repair the A53T mutation in PD patient-derived iPSCs. In the
absence of drug selection, Soldner et al. delivered ZFNs targeting
the G209A mutation in exon three of SNCA and a wild-type
sequence-containing donor template carrying ~1 kb homology
sequence flanking the targeting site. One of 240 single-cell-derived
clones contained the correctly repaired nucleotide at position 209
of SCNA. Subsequent pluripotency marker expression analysis and
teratoma formation assays indicated that the genetically corrected
clone maintained pluripotency. Moreover, cells from the clone were
able to differentiate into tyrosine hydroxylase-expressing dopaminergic neurons that no longer expressed the mutant transcript.
This elegant set of experiments demonstrates the utility in
combining genome editing strategies with iPSC technology to
interrogate disease-causing mutations and therapeutically correct
them in vitro.
TALENs were used to modify the murine coagulation factor VIII
(F8) locus in iPSCs (Park et al., 2014a). Mutations in this locus in
humans cause one of the most common bleeding disorders, Hemophilia A. Park et al. engineered TALEN pairs targeted to intron 1
of the murine F8 gene and electroporated plasmids encoding the
most efficient pair into wild-type iPSCs. After clonal selection and
PCR analysis, the researchers recovered six of 432 (1.4%) colonies
with a 140 kbp inversion resulting from non-allelic homologous
recombination (HR), thus creating model cell lines containing a
common mutation in patients with Hemophilia A. Expression
analysis in the inversion clones indicated loss of F8 mRNA and
protein. Importantly, Park and colleagues were able to restore F8
expression through inducing reversion of the 140 kbp segment by
delivering the same TALEN pair to the mutant clones (Park et al.,
2014a). Thus, the researchers demonstrated the use of genome
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
editing technologies to both model and correct genetic diseases in
the culture dish.
Recently, Wu and colleagues utilized the CRISPR-Cas9 system
to correct a dominant mutation in the murine Crygc gene e mutations in which cause cataracts (Wu et al., 2013). Co-injection of
Cas9 mRNA and an sgRNA targeting the mutant allele with one of
two oligonucleotide repair templates into Crygc mutant embryos
yielded HDR-mediated repair in 5 of 29 (Oligo-1; 17.2%) and 4 of
27 (Oligo-2; 14.8%) live-born pups. All mice that carried the corrected allele induced by HDR were free of cataracts and the lenses
were histologically normal compared to non-rescued mutant mice
(Wu et al., 2013). These results demonstrated that the CRISPR-Cas9
system could be used to correct genetic disease phenotypes
in vivo.
While the combination of genome editing strategies with iPSC
technology represents a powerful avenue by which to investigate
the pathophysiology of inherited retinal dystrophies as well as efficacy of treatment, there is concern that DSB induction at off-target
sites will create adverse effects such as genotoxicity. Indeed, the
CRISPR/Cas9 system can tolerate up to five mismatches in the
sgRNA target site (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al.,
2013). In addition to determining efficacy of therapeutic strategies,
evaluation of undesirable off-target modifications will be essential
when employing genome editing in the clinic. The majority of
bioinformatically predicted off-target modifications can be
assessed through PCR amplification and Surveyor nuclease digestion (Fig. 5). Surveyor nuclease is sensitive enough to detect from
about three percent modifications at a locus (Qiu et al., 2004),
however, genome wide analysis using deep sequencing technologies allows for thorough assessment of safety in modified cells.
Several groups have performed off-target analyses using these
methods and results indicate that modification at unintended loci
occurs very rarely (Duan et al., 2014; Güell et al., 2014; Li et al.,
2013; Soldner et al., 2011; Wang et al., 2013; Wu et al., 2013).
In addition, Wu and colleagues evaluated off-target mutations in
CRISPR-Cas9 modified mice via DNA sequencing of PCR products
from ten potential off-target sites in twelve mice. No mutations
Fig. 5. Off-target mutation analysis in cells treated with CRISPR/Cas. A) Schematic
representation of the EMX1 locus. A previously described sgRNA targeting just upstream of exon 3 (red arrow) of EMX1 (sg5) (Ran et al., 2013a) was delivered with the
wild-type Cas9 nuclease to HEK293T cells. B) The Optimized CRISPR Design tool
(http://crispr.mit.edu) was used to bioinformatically predict the off-target sites (Cong
et al., 2013). The top sites are shown in the table. Bolded letters indicate base
changes from the sgRNA, sg5. C) Surveyor nuclease activity at the on-target (sg5) and
four off-target (OT) sites in duplicate. Genomic DNA from cells treated with sg5 (þ) or
untreated controls () were subjected to Surveyor nuclease detection analysis (Ran
et al., 2013b). sgRNA: small guide RNA, PAM: protospacer adjacent motif, OT: off-target.
11
were recovered in ten of the twelve mice evaluated. Of the
remaining two, only one site carried off-target modifications (Wu
et al., 2013).
To address the issue of undesirable off-target modifications,
investigators employ a modified Cas9 nuclease (Ran et al., 2013a).
The modified Cas9 carries a catalytic amino acid substitution
(D10A) in the conserved RuvC nuclease domain converting the
enzyme to a ‘nickase’, i.e. Cas9 cleaves only the strand noncomplementary to the guide RNA. Single-stranded nicks are repaired by
the base excision repair pathway (BER) (Dianov and Hübscher,
2013), and thus maintain genome integrity. By delivering the
Cas9-nickase guided by a pair of sgRNAs targeting opposite strands
of the target locus, the nicking enzyme creates DSBs with minimal
off-target modification. Ran and colleagues recently demonstrated
a 50e1500-fold reduction in off-target cleavage using this
approach. Importantly, employing the nickase strategy maintains
on-target activity (Ran et al., 2013a). Similarly, shortening the
sgRNAs can reduce off-target activity. Fu et al. showed that sgRNAs
of 17 or 19 nucleotides in length reduced off-target activity by as
much as 5000-fold in some cases (Fu et al., 2014). Again, these
truncated guides showed comparable on-target activity. Employing
either truncated guides or paired nickases, or both strategies in
concert can greatly reduce undesired off-target effects. These
exciting technologies, in addition to future developments in
genome editing, will be essential in allowing immunologicallymatched and genetically wild-type cell replacement in heritable
forms of blindness.
3.3. iPSCs for testing of therapeutic efficacy
Although human stem cells represent an exciting new frontier
for treatment of inherited blinding diseases, a sobering aspect of
this otherwise exciting progress is the sluggish pace at which the
few therapies that are available today have moved from “proof-ofconcept” stages in animals to a fully-approved treatment available
to patients who need them.
An example of this is the substantial amount of time it took for
RPE65 gene replacement therapy to become a reality for patients
afflicted with RPE65-associated LCA. Efficacy of RPE65 gene
replacement was demonstrated in a canine model in 2001 (Acland
et al., 2001), and over thirteen years later, fewer than 100 patients
have been treated world-wide and the Phase III clinical trial is still
ongoing. This raises the possibility that some diseases might be so
rare in the population that it is not economically logical and feasible
to bring a treatment through the regulatory gauntlet and into
clinical availability. One possible solution for disorders that are so
rare that they are below the commercial viability economic
threshold is to use autologous patient-derived cells to test the
molecular efficacy of a drug or viral-mediated gene therapy.
Although the optimal dosages of therapies may not be identical in
culture and in vivo, demonstrating efficacy and a range of tolerated
and effective doses in patient-specific cells will be an important
step forward. We envision that the identified therapies can then be
delivered to one eye in a compassionate use manner. Patients
treated in this way can be monitored at intervals using conventional clinical measures such as visual acuity, Goldmann perimetry,
ganzfeld electroretinography and optical coherence tomography. In
the event treated eyes fare worse than untreated ones, the
compassionate use treatment can be stopped until the reason for
the poorer outcome can be identified and an improved treatment
can be developed. When treated eyes display noticeable improvement in vision than untreated eyes over time, the treatment can
then be offered to new patients and the second eyes of the initial
patients can also receive treatment. In adopting a treatment
approach like this, a series of increasingly predictable, reusable
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
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L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
parts can be assembled and used to devise a compassionate use
therapy for retinal disorders, regardless of their rarity.
cells will enhance the efficiency of drug discovery and decrease the
time lag between compound discovery and Phase I clinical trials.
3.4. iPSCs and drug discovery
3.5. Retinal transplantation and cellular replacement
For translational applications like discovering novel pharmacotherapeutics, human stem cell-based assays represent a physiologically relevant and high-throughput means to assess efficacy and
toxicity early on in the drug development pipeline. Human stem
cells are a means for expediting early proof-of-concept studies for
potential drug candidates. As indicated above, retinal cells derived
from iPSCs are faithful representatives of their in vivo counterparts
(Tucker et al., 2013b). High-throughput drug screens of differentiated iPSC-derived cells have been suggested as a means for drug
discovery and personalized medicine (Desbordes and Studer, 2013;
Inoue and Yamanaka, 2011). Drug screening using extra-ocular
iPSC-derived cells from patients with rare diseases has shown
excellent promise. Screens have been performed on iPSC-derived
cardiomyocytes (Cerignoli et al., 2012; Mordwinkin et al., 2013),
forebrain neurons (Luo et al., 2013; Yahata et al., 2011), motor
neurons (Egawa et al., 2012), and hepatocytes (Kim et al., 2013), to
name a few examples.
Combining the power of differentiated ocular cells with highthroughput screens will likely prove effective for discovering new
pharmacologic treatments for delaying the progression of retinal
degenerative diseases. High-throughput drug screens will need to
be highly organized and well thought out, however, if they are to
successfully identify reliable drug treatment candidates. Screening
assays will need to be multiplexed to simultaneously assess the
proliferation, morphology, differentiation state, viability and function of specific iPSC-derived retinal cell types that would enable the
monitoring and testing of disease-relevant phenotypes (Wright
et al., 2013).
To date there have been a limited number of reports looking at
the effects of drugs on iPSC-derived retinal cells. David Gamm and
collaborators reported the restoration of ornithine aminotransferase activity in iPSC-derived RPE from a patient with gyrate atrophy following treatment with vitamin B6 (Meyer et al., 2011a). It
has been known for some time that vitamin B6 is beneficial for
patients with gyrate atrophy. However, the patient of interest in
this study was previously unresponsive to vitamin B6 supplementation. This study highlighted the use of iPSC-derived RPE to test
the specific cellular population targeted in gyrate atrophy to more
accurately test vitamin B6 efficacy.
In another study, the lab of Masayo Takahashi generated patientspecific iPSC-derived rod photoreceptor-like cells harboring mutations in several genes that cause retinitis pigmentosa like RP1,
PRPH2, RHODOPSIN, and RP9 (Jin et al., 2011). This study showed
that diseased RHODOPSIN-positive iPSC-derived retinal cells perished after 120e150 days in the culture dish. The authors then
treated cells with 3 antioxidant vitamins, a-tocopherol, ascorbic
acid and b-carotene. They observed that cells treated with atocopherol led to a statistically significant increase in survival of
diseased iPSC-derived photoreceptor lines, providing proof-ofprincipal for drug screening of iPSC-derived retinal cells.
IPSC technology offers an unlimited source of disease- and
patient-specific material for automated, high-throughput drug
screening systems to screen libraries of drug compounds. As evidence for the efficacy of this strategy, work from the laboratory of
Donald Zack, which utilized a high-throughput RNA interference
screen and primary mouse retinal ganglion cells, demonstrated
that large compound libraries could be effectively screened in a
rapid fashion to identify novel therapeutics that promote retinal
ganglion cell survival (Welsbie et al., 2013). Ultimately, replacing
primary neurons in these experiments with iPSC-derived retinal
Cell replacement therapy is simply defined as replacing dead or
damaged cells with healthy cells to restore the function that a
specific cell population provides within a tissue. The retina is a good
candidate for cell replacement therapies for several reasons.
Compared to other constituents of the central nervous system
(CNS), the posterior retina is very accessible to therapeutic delivery
through subretinal injection, a standard technique that can be
performed by most vitreoretinal surgeons. Clinical diagnosis and
post-intervention examining of the retina are also feasible
compared to other regions of the CNS due to the transparency of
the cornea, lens, and intraocular media.
However, there is a longstanding argument amongst clinicians
and vision scientists pertaining to the following key questions: 1)
What is the optimal stem cell source for production of outer retinal
cells?; 2) What is the optimal stage of differentiation for posttransplant survival, integration and cellular function?; and 3)
How will the local diseased environment respond to transplanted
cells immunologically?
1) What is the optimal stem cell source for production of outer
retinal cells?
Retinal progenitor cells (RPCs) have been investigated as a potential option for retinal cell replacement therapy. These cells can
be isolated from early embryonic tissue and give rise to ganglion,
amacrine, horizontal, retinal pigmented epithelium, and photoreceptor cells (Baranov et al., 2014; Chatoo et al., 2010; Hafler et al.,
2012; Jadhav et al., 2006; Luo et al., 2014; Rompani and Cepko,
2008; Rowan et al., 2004; Schmitt et al., 2009). Subretinal transplantation of GFP-positive RPCs into retinal degenerative mouse
models results in migration of the transplanted cells into the outer
nuclear layer, differentiation into immunohistochemically identifiable rod photoreceptor cells and functional rescue of blindness in
the form of improved pupillary light responses (Klassen et al.,
2004). Likewise, transplantation of human RPCs into the subretinal space of the RCS rat was shown to slow disease progression
(Luo et al., 2014). As promising as RPCs are, as indicated above,
these cells are obtained from post-mortem embryonic globes and
as such are limited in number, rife with ethical concerns and are
immunologically dissimilar from the transplant recipient.
In light of this, the two most promising stem cell types for human retinal transplantation are human embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs). As indicated above, ESCs
are harvested from developing embryos approximately 4e5 days
after fertilization just prior to implantation (Thomson et al., 1998),
whereas iPSCs can be generated by reprogramming various cell
types with cocktails of transcription factors, such as adult dermal
fibroblasts via viral transduction of the transcription factors Oct4,
Sox2, Klf4, and c-Myc. Both ESCs and iPSCs can be expanded to yield
sufficient cell numbers for clinical applications and both have the
capacity to generate all of the various cell types of the retina. To
date, differentiation protocols capable of generating photoreceptor
precursor cells from both ESCs and iPSCs have been developed
(Ikeda et al., 2005; Lamba et al., 2006; Meyer et al., 2011b, 2009;
Nakano et al., 2012; Osakada et al., 2008, 2009a,b; Tucker et al.,
2013a, 2011a).
Like RPCs, ESCs and iPSCs each possess their own pros and cons.
For example, ESCs could represent a ‘universal’ donor stem cell
population, but the harvesting and use of ESCs is highly controversial from an ethical point of view, and like RPCs, ESCs would be
an allogenic transplanted cell population, as donor and recipient
would not be immunologically matched. This situation could have
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
negative consequences within the retina following transplantation,
including but not limited to, rejection of the transplanted cells and
an unwanted inflammatory response in the eye. On the other hand,
iPSCs represent an autologous donor population because they are
generated from somatic cells isolated from the patient for which
they are intended (e.g. skin-derived fibroblasts or keratinocytes).
However, as we will describe further below, the retinal immune
environment may be more complicated, and even this cell type may
require post-transplant immune modulation. A drawback to the use
of autologous iPSCs is that since they are generated on a patientspecific basis, they will take much longer to produce and as they
will be produced ‘as needed’ they will require significantly more
manpower and resources to meet clinical demands. Additionally,
depending on the patients' original disease-causing mutation, genetic correction may also be required. Although time will tell which
of these cell types will be optimal for therapeutic cell replacement,
we have chosen to hedge our bets with the autologous iPSC-based
strategy.
2) What is the optimal stage of differentiation for post-transplant
survival, integration and cellular function?
The next big question after deciding which stem cell source to
utilize is at what stage of differentiation should cells be in order to
achieve optimal levels of post-transplant survival, integration and
function? For photoreceptor cell transplantation, the post-mitotic
photoreceptor precursor cell has been shown to be at the optimal
stage of development for retinal transplantation (i.e. it possesses
the greatest capacity for cellular integration and restoration of
retinal structure) (MacLaren et al., 2006). Unfortunately, like RPCs,
in humans these cells are isolated during late embryonic development, which raises serious ethical and practical concerns (i.e. it
would be difficult to obtain sufficient numbers of viable cells for
transplantation). Fortunately, both ESCs and iPSCs have all been
shown to give rise to photoreceptor precursor cells postdifferentiation and following transplantation from new functional
photoreceptors (Bartsch et al., 2008; Gonzalez-Cordero et al., 2013;
Lamba et al., 2009; Tucker et al., 2013b, 2011a; West et al., 2012; Yao
et al., 2011). Of the differentiation paradigms used, ours was
developed by combining aspects of previously published differentiation strategies (Ikeda et al., 2005; Lamba et al., 2006; Osakada
et al., 2008) which take into account the role of bone morphogenic protein (BMP) and Wnt signaling pathway inhibition in
neuroectodermal development (Anderson et al., 2002; Lamb et al.,
1993; Mukhopadhyay et al., 2001), the role of IGF-1 in anterior
neural/eye field development (Pera et al., 2001), and Notch
pathway inhibition in photoreceptor development (Jadhav et al.,
2006). Using this differentiation program, iPSCs readily form
embryoid bodies within days and early retinal progenitor cells
13
(RPCs) within weeks. These iPSC-derived RPCs express the retinalspecific markers OTX2, SIX6, VSX2, RX and CRX. Following 2e3
more months, these cells further differentiate into eyecup like
structures with post-mitotic photoreceptor precursor cells that
express the photoreceptor-specific markers recoverin and
rhodopsin (Fig. 6), phosducin, red/green-opsin and blue-opsin
(Tucker et al., 2013a,b, 2011a,b). Although promising for
photoreceptor-only diseases, the optimal developmental target is
yet to be determined for RPE and choroidal endothelial cells. As
such, in situations where multiple cell types are required further
experimentation is needed. In disease states such as these, autologous cell sources will likely be critical.
Regardless of stem cell source and differentiation stage, differentiated retinal cell types will need to be purified from other cell
types present in the culture before transplantation in the clinic.
Generating and purifying photoreceptors or choroidal endothelial
cells in great enough numbers presents a challenge. Lamba and
colleagues purified photoreceptors generated from human iPS cells
using fluorescence activated cell sorting (FACS) after transduction
with a lentivirus expressing GFP from a photoreceptor-specific
regulatory elements. The purified photoreceptors integrated into
mouse retina and expressed photoreceptor-specific markers Otx2,
recoverin, and rhodopsin, supporting the proof-of-principle that
differentiated photoreceptor precursors can be purified and
transplanted successfully (Lamba et al., 2010). Genetically modified
photoreceptors, however, would not be desirable for treatment,
thus alternate methods of purification should be explored.
Lakowski et al. isolated photoreceptor precursor cells from developing murine retinae using the photoreceptor cell surface antigens
Cd24a and Nt5e and FACS. The researchers transplanted the
selected photoreceptor precursor cell population into wild-type or
degenerative murine host eyes (Crb1/ or Prph2/). The selected
cells migrated into the outer nuclear layer, acquired photoreceptor
morphology and expressed outer segment markers at an 18-fold
higher integration efficiency than that of unsorted cells (Lakowski
et al., 2011). These important experiments indicate the feasibility
of using cell selection strategies employing antibodies recognizing
cell surface markers to purify photoreceptors for transplantation.
One can envision a similar strategy for use with choroidal endothelial cells.
3) How will the local diseased environment respond to transplanted cells immunologically?
3.6. Retinal immune landscape
The eye has long been believed to be an organ that boasts
“immune privilege” from the body's surrounding immune system.
Fig. 6. IPSC-derived photoreceptors express photoreceptor-specific markers. A) Immunofluorescent labeling of recoverin (green), tubulin (red) and DAPI (blue) in differentiated
iPSC-derived photoreceptor cultures. Inset shows a magnified view of recoverin-positive photoreceptor-like cells. B) High magnification example of a differentiated neural rosette
that is comprised of photoreceptor-like cells that express the photoreceptor-specific markers, recoverin (green) and rhodopsin (red). Scale bar ¼ 400 mm.
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
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L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
It is believed that this “immune privilege” exists for the purpose of
protection from unwanted inflammatory responses and helps to
maintain the intraocular microenvironment. The first description of
“ocular immune privilege” was made by Dutch ophthalmologist J.C.
van Dooremaals in 1873 (Van Dooremaal, 1873). Dr. van Dooremaals observed that tumor cells injected into the anterior chamber of the eye survived, proliferated and formed intraocular tumors
(Van Dooremaal, 1873). Many years later it was demonstrated that
the eye is not entirely “immune privileged,” but the response to
intraocular antigens is highly controlled and specialized, a phenomenon that became known as anterior chamber-associated immune deviation (ACAID) (Kaplan and Streilein, 1977; Stein-Streilein
and Streilein, 2002; Streilein, 1987; Streilein and Niederkorn, 1981).
ACAID is the process by which the eye communicates with the
surrounding immune system via the spleen to tolerate antigen
presenting cells in the eye and thus avoid a delayed, heightened
systemic immune response (Stein-Streilein and Streilein, 2002;
Streilein and Niederkorn, 1981). A common clinical example of
the tolerance allowed due to ACAID is the allogenic transplantation
of avascular corneal grafts, a highly successful surgery that requires
no immunological matching between donor and recipient, merely
local immunomodulation via topical drops. Although the retina
does not possess the same level of immune privilege as the anterior
chamber, the blood-retinal barrier (BRB) affords some protection.
The BRB acts as a tight barrier between the retina and that of the
systemic circulation, namely circulating immunomodulatory and
effector leukocytes. The BRB actually consists of two separate
anatomical sites: 1) the endothelial cells, pericytes, and glia of the
non-fenestrated inner retinal vasculature and 2) the tight junctioncontaining, monolayered RPE on Bruch's membrane between the
fenestrated choroidal vasculature and outer retinal vasculature
(Crane and Liversidge, 2008). In a normal, healthy retina, though
not immune privileged, the BRB provides the retina with an immune advantage, keeping it isolated from the surrounding immune
system. However, when the retina is damaged, particularly if the
integrity of the RPE and choroidal vasculature is compromised,
circulating immunomodulatory factors have easier access to the
neural retina. In most degenerative retinal diseases, the RPE is
significantly injured. Using human donor eyes from patients with
various retinal degenerative diseases like ABCA4-associated Stargardt disease, Best disease and AMD, we have shown that loss of
RPE is a common characteristic among these disorders (Mullins
et al., 2012, 2007). Support for the BRB playing a major role in
conferring protection from the systemic immune system lies in
evidence that the subretinal space becomes much more proinflammatory in the face of photoreceptor and RPE degeneration
(Chinnery et al., 2012; Mullins et al., 2012; Rutar et al., 2010). There
are also reports that inflammation is heavily involved in the pathogenesis of AMD (Ambati et al., 2013; Anand et al., 2003; Tarallo
et al., 2012; Whitcup et al., 2013). Furthermore, it has been suggested that the immune system may be critical in the pathogenesis
of Batten disease. Cellular infiltrates have been observed in the
vitreous cavity of molecularly-confirmed Batten patients (Fig. 7),
and these patients also possess circulating autoantibodies, particularly directed against GAD65 (Chattopadhyay et al., 2002; Drack
et al., 2014; Pearce et al., 2004). Immunosuppressive agents have
demonstrated efficacy in slowing the progression of CLN3-associated Batten disease in a genetic mouse model and a human patient
(Drack et al., 2014; Seehafer et al., 2011).
The above examples of how loss of BRB integrity in numerous
retinal degenerative diseases leaves the retina vulnerable to
circulating immune cells and immunomodulatory factors are the
reason we believe that the most promising cellular population for
future use in human cellular transplantation trials is that of autologous, immunologically-matched patient-derived iPSCs. While
ESCs may be more readily available in a shorter timeframe, the fact
that they are not immunologically matched between donor and
recipient makes it highly likely that they will incite an acute immune response, leading to rejection of the transplanted cells and
perhaps further damage to an already diseased eye. This concern is
of particular importance if the patient receiving ESCs still has some
functional vision remaining. That being said, ESCs would likely still
be a good option for transplantation and would be tolerated to a
better degree in early-stage diseased retinas in which the RPE is
relatively undamaged and BRB integrity remains intact. While using autologous iPSCs will likely reduce the incidence of immunological reactions and rejection of transplanted cells, the degree to
which immunologic matching will be required for the survival,
integration, function, and longevity of stem cell-based retinal
transplants is a critical unanswered question in the field. Definitively testing this hypothesis will allow us to conserve societal resources (if patient-specific cell sources prove not to be required for
successful therapeutic transplantation); or, prevent harmful immune activation in visually impaired patients (if our studies show
that immune matching of the transplant is necessary).
4. Summary
Patient-specific induced pluripotent stem cells have emerged as
a promising tool for identification and interrogation of diseasecausing mutations, testing efficacy of novel therapeutics, and as a
cell source for autologous retinal cell replacement. As depicted in
Fig. 8, the key to patient-specific therapeutic development is
thorough phenotypic assessment and accurate clinical diagnosis of
Fig. 7. Cellular infiltrates in the vitreous cavity of a patient with CLN3-associated Batten disease. A) Fundus photograph of the right eye of a child with molecularly-confirmed CLN3associated Batten disease showing bulls eye maculopathy and granularity of the fovea, both common presentations in eyes afflicted with Batten disease. B) Slit lamp photograph of
the same eye in (A) and a magnified view of cellular infiltrates present throughout the vitreous cavity (outlined in red box).
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
15
Fig. 8. Schematic diagram depicting the strategy for use of iPSCs for the interrogation and development of patient-specific therapeutics for retinal degenerative diseases.
inherited retinal disease, taking into account patient and family
history, high-resolution retinal imaging (OCT and fundoscopy), visual field and electroretinographic analysis, followed by molecular
confirmation (genotyping) of disease-causing mutations via
sequencing of patient DNA (from blood). A skin biopsy obtained
during the initial clinical visit can be used for generation of autologous patient-derived iPSCs. IPSCs and iPSC-derived retinal cells
can then be used to interrogate disease pathophysiology, develop
drug, genome editing, gene augmentation and cell-based therapies,
as well as to discover novel mutations that cannot be identified
using standard sequencing methods, such as those located in promoter or intronic regions of the gene. Finally, strategies developed
and tested against patient-specific disease mutations and phenotypes can then be utilized for human gene and cell replacement
therapies.
5. Future directions
Many exciting developments in the field of iPSC technology
present an excellent opportunity to treat inherited retinal degenerative diseases and ultimately improve patients' lives. IPSCs can be
employed to investigate and determine disease mechanisms from
individual patients as well as determining pathogenicity of novel
mutations. Knowledge gained from these experiments can be used
Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal
degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002
16
L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21
to develop and test drug- and gene-based therapies. Transplantation of gene-corrected patient-specific iPSC-derived retinal
neurons will treat individuals with advanced-stage retinal degenerative disease. These approaches will be expedited through production of clinical-grade reagents (i.e. patient-specific iPSC-derived
retinal photoreceptor cell lines and viral vectors) in dedicated
current good manufacturing production (cGMP) facilities. Clinicalgrade stem cells and therapeutic vectors can be utilized in a
compassionate use manner to treat aggressive neurodegenerative
blinding diseases to pave the way for treatments of other rare
inherited retinal dystrophies that fall below the commercial
viability threshold. For patients with vision threatening diseases,
for the first time there is cause for real hope.
Acknowledgments
We would like to graciously thank the following funding organizations: NIH Directors New Innovator Award 1-DP2-OD00748301; NEI EY017451; HHMI; Foundation Fighting Blindness; Stephen
A. Wynn Foundation; Grousbeck Family Foundation; Leo, Jacques &
Marion Hauser Family Vision Restoration Fund; NIH F32 EY022834.
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