JUNIPER PUBLISHERS- JOJ Ophthalmology
Stem cells provide a promising tool for treating
retinal diseases and injury. Early work focused on embryonic stem cells
(ESC). The development of induced pluripotent stem cells (iPSC)
alleviates some of the ethical concerns with ESC and the need for
immunosuppression. Stem cell-derived retinal pigment epithelial cells
(RPE) are comparable to native RPE; and stem cell-derived retinal
organoids self-organize into laminated structures that bear some
resemblance to the neurosensory retina. Questions remain regarding
genetic and epigenetic variability among different stem cell lines,
especially iPSC lines. The challenge is in understanding the
significance of this variability for transplant and how to control such
variability. Transplantation of stem cell-derived RPE and retinal
progenitor cells has been tested in both animal models and humans. The
cells integrated into the recipient with possible rescue of visual
function. These findings encourage researchers to develop refined
culture and delivery methods that would increase integration with the
host and sustain long-term visual function.
Introduction
Since the beginning of stem cell research,
pluripotent cells were seen as a promising tool for tissue regeneration
and transplantation. Widely known, stem cells have the ability to
differentiate into one or more mature cell types or continue to renew
themselves. These properties make stem cells a potential source for
sustained supply of tissue for transplantation. There is growing
interest in developing stem cell therapies for neurodegenerative
diseases, such as Alzheimer and amyotrophic lateral sclerosis (ALS),
with the aim of replacing diseased tissue [1,2]. Similarly, research on developing replacement tissues for retinal degeneration has gathered momentum as well.
Sources of Stem Cell-Derived Retinal Cells
In many retinal diseases, such as age-related macular
degeneration (AMD) and retinitis pigmentosa, there is degeneration of
both the retinal pigment epithelium (RPE) and photoreceptors resulting
in vision loss. The native retina, being a neural tissue, has little
ability to regenerate. Therefore, transplanted tissue needs to replace
lost tissue, continue to survive, and integrate functionally into the
host retina. Several sources of stem cells have been used for
regenerating retinal tissue; the most extensively studied are human
embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC).
Additionally, researchers have developed and continue to improve
different methods of differentiating stem cells into retinal cells.
Embryonic stem cells
Embryonic stem cells (ESC) are pluripotent stem cells derived from blastocysts in early development [3,4].
In stem cell biology, they are considered the gold-standard for
comparing pluripotency, and genetic and epigenetic characteristics. The
ethical concerns of human ESC have been much debated. Nevertheless, ESC
allowed early successful differentiation of retinal cells in culture.
hESC-derived RPE demonstrated robust pigmentation, and exhibited similar
morphology and gene expression as human fetal RPE [5-8]. These RPE cells also developed appropriate functional characteristics, such as phagocytosis of shed outer segments [9,10].
Scanning across the literature, there are now various differentiation
methods used for generating retinal cells. Some methods involve directed
differentiation using small molecules and growth factors, and others
allow stem cells to spontaneously differentiate into RPE in specialized
media [9,10].
The time taken for retinal cells to differentiate can vary depending on
the protocol and the desired cell type. Neuroretinal precursor cells,
for example, can appear as early as day 10 in culture, while pigmented
cells can take 6-8 weeks to appear [11,12].
Researchers continue to improve methods to generate retinal cells
comparable to native retina in order to study retinal development and to
generate tissue for transplantation.
Induced pluripostent stem cells (iPSC)
iPSCs are pluripotent stem cells that are derived
from adult cells. Skin fibroblasts and peripheral blood cells are
commonly used to generate iPSC. The Yamanaka group were the first to
describe this reprogramming by introducing four transcription factors
Oct3/4, Sox2, Klf4, and c-Myc, known as the “Yamanaka factors”. [13,14]
iPSCs also have been successfully differentiated into retinal cells.
iPSC-derived RPE can attain appropriate barrier function including
proper distribution of membrane NaK-ATPase, polarized secretion of VEGF
and similar membrane potential as native RPE [15].
Retinal progenitor cells and photoreceptors derived from iPSC also
exhibit similar gene expression patterns as those derived from ESC,
although there can be variation in the timing of differentiation [16].
iPSC can be a source of unlimited supply of
regenerated tissue for studying development and for transplantation. One
major foreseeable advantage of iPSC over ESC is the issue of immune
histocompatibility. iPSC derived from a patient’s adult cells would not
cause immune rejection when transplanted into the same person. In
practice, not every iPSC line can successfully differentiate into the
desired cell type. There is in fact variability among iPSC lines. Some
researchers ascribe the cause of variability to differences in
reprogramming techniques and lab environment; others propose that iPSC
have different epigenetic markers either due to the reprogramming
procedure or epigenetic memory of the original adult cell [17-19]. However, there is controversy over how much epigenetic aberrancies contribute to the variability seen among iPSC lines [20].
The ultimate question is how cellular variability
affects the safety of iPSC-derived cells for transplantation. There is a
need for defining standards not only to evaluate iPSC lines but also
the differentiated cells derived from iPSC. Miyagishima et al. [21]
proposed a system of authenticating iPSC-derived RPE: in addition to
assessing gene expression and morphology, they also assessed cellular
calcium flux, membrane electrophysiology and fluid transport in
comparison to human fetal RPE [22].
Rigorous testing and characterization is needed to increase the safety
and integrity of retinal tissue selected for transplantation.
Retinal Transplantation
Transplantation of stem-cell derived retinal cells in
animal models has presented positive results in visual improvement.
Human clinical trials demonstrated good long-term safety of
transplantation [22,23].
There are several ongoing clinical trials using stem-cell derived
retinal cells for retinal diseases. The goals of transplantation are to
replenish and rescue degenerating cells, re-establish neural
connectivity within the retina, and improve visual acuity.
RPE transplantation
Overall, more translational studies have been done
using stem-cell derived RPE than with stem-cell derived neuroretinal
cells. Transplantation studies commonly use rodent models of retinal
degeneration. A widely used model, for example, is the Royal College of
Surgeons (RCS) rat, which has a mutation in MERTK gene and models
autosomal recessive retinitis pigmentosa [24].
Transplantation of ESC-derived and iPSC-derived RPE in rodents with
retinal degeneration resulted in more photoreceptor survival compared to
non-transplanted animals. The photoreceptor layer was thicker at the
transplant site compared to control [9,11,25-27].Transplanted
RPE also promoted better visual function, measured by electroretinogram
or optokinetic testing, compared to control animals [9,11,26].
The exact mechanism of photoreceptor rescue is not entirely elucidated.
Given that the transplanted RPE does not always restore the outer
blood-retinal barrier, one can postulate that trophic factors secreted
by the RPE and the phagocytosis of photoreceptor outer segments may
mediate the protective effects on the degenerating photoreceptors.
One major challenge from the studies mentioned above is long-term graft survival and visual improvement. In Carr et al. [10] implanted iPSC-RPE cells were eventually lost in the host retina at 13 weeks after transplant [26].
The mice interestingly retained improved visual function even when
transplanted cells were not present. However, it is unknown whether this
visual preservation can be sustained for longer. In the Idelson et al. [9]
study, for example, the increased electroretinogram signal in
transplanted animals eventually diminished at later time points (19
weeks). These results are proof-of-concept for using stem-cell derived
retinal tissue to improve vision in retinal diseases. However, they also
highlight limitations and challenges that need to be overcome to
improve effectiveness of transplantation. The route of transplantation
is seen as an area for improvement. In earlier transplant studies, a
bolus of cells suspended in solution was injected into the subretinal
space. This delivery method limits the ability of the transplanted RPE
to re-organize into a functional monolayer; perhaps relatedly, cell
survival from bolus injections is low [28].
Active research now focuses on transplanting sheets of RPE grown of
various scaffolds to promote increased graft survival in the recipient [29,30].
In 2015, human clinic trial results for hESC-RPE transplantation in two retinal diseases were reported [23].
The trials were phase I/II with primary outcomes of safety and
tolerability. The grafted cells were well tolerated without evidence of
aberrant growth or serious side effects. When visual acuity was measured
at 6 months after transplant, 6 out of the 9 AMD patients showed modest
improvement from baseline and 3 out of 8 Stargardt’s macular dystrophy
patients showed similar improvement. The other patients had stable or
decreased visual acuity. The study demonstrated the safety of stem-cell
derived retinal transplantation in human patients. Other clinical trials
are underway to assess different types of stem-cell derived retinal
tissue, different methods of delivery, and in different retinal
diseases.
Photoreceptor transplantation
Efforts to replace diseased photoreceptors have
involved transplantation of retinal progenitor cells (RPC).
Understandably, mature neural retina is more challenging to
differentiate in culture, given its complex interconnected laminations.
However, RPC have been successfully grown from stem cells and
transplanted into animal models with the hope that these progenitor
cells can continue differentiation into mature retinal cells in the
host.
Several groups developed methods of differentiating
stem cells into three-dimensional, spherical organoids composed of
retinal progenitor cells [31-34].
The organoids (referred to in the literature as optic vesicles)
contained cells that expressed developmental markers for photoreceptor,
amacrine, horizontal and ganglion cells; with time in culture, the cells
within optic vesicles self-organize into crude laminations [32,35].
One group demonstrated electrical excitability in these optic vesicles,
indicating functional synaptic connectivity among the cells. The
generation of these stem cell-derived optic vesicles offers a method of
increasing production efficiency of neural retinal tissue for
transplantation. However, the spherical geometry of the organoids makes
them unsuitable for implantation, because they fail to flatten and
simultaneously interact with the RPE and neurosensory retina. As models
of retinal differentiation, they should prove valuable for studying the
mechanisms of retinal disease and potential medical therapies.
Transplantation with immature RPC also has had
positive results in animal models. Transplanted stem cell-derived
retinal precursor cells migrated into and integrated structurally with
the host retina, showing synaptic interaction with the host [12,36-38].
Furthermore, better visual function was assessed by optokinetic
testing, electroretinogram, and visual cortex activity in transplanted
animals compared to control [37,39]
Like stem cell-derived RPE, the stem cell-derived neural retinal cells
are well tolerated in the recipient. However, there is still little data
on long-term survival of these stem cell derived-retinal progenitor
cells, and whether vision can also be rescued long-term. These
encouraging results highlight the need for more validation studies in
preclinical models.
Conclusion
Researchers have successfully differentiated retinal
cells from ESC and iPSC. Retinal culture systems, such as the
three-dimensional organoids, allow the study of retinal development,
mechanisms of disease, and provide tissue for transplantation in retinal
diseases. There is continued modification and optimization of these
differentiation methods. Both stem cell-derived RPE and retinal
progenitor cells have been transplanted in animal models and exhibited
graft survival and possible visual improvement. For human patients,
early phase trials demonstrated good tolerability of transplantation.
More clinical studies are needed to validate the efficiency of retinal
transplantation.
Grant Support
Leir Foundation, Newman’s Own Foundation.
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