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Master’s and Doctoral Projects
2004
Limbal stem cell transplantation for the restoration
of vision after corneal damage : a literature review
Kimberly Diane Hills
Medical College of Ohio
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Hills, Kimberly Diane, "Limbal stem cell transplantation for the restoration of vision after corneal damage : a literature review" (2004).
Master’s and Doctoral Projects. Paper 331.
http://utdr.utoledo.edu/graduate-projects/331
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Limbal Stem Cell Transplantation for the Restoration of Vision
After Corneal Damage: A Literature Review
Kimberly Diane Hills
Medical College of Ohio
2004
ii
Dedication
This paper is dedicated to my husband, Aaron, who has worked so hard to support us
so I could finish school, who has always been there when I needed him, and has loved
me more than I thought possible; To my parents, Scott and Karen Sturgis, who have
always shown unconditional love and support; and to Magoon, who was always so
proud of me – I miss you.
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Acknowledgements
Special thanks to my academic advisor, Jolene Miller, MLS, who was willing to work
with me and has given me great advice.
Special thanks to Michael May and those like him who are willing to be pioneers in new
scientific endeavors so that others may benefit.
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Table of Contents
Introduction ..................................................................................................................... 1
Stem Cell Theory............................................................................................................. 4
Anatomy and Physiology of the Visual System ............................................................... 7
Cortical Plasticity ........................................................................................................... 17
Stem Cell Transplantation to Reverse Blindness due to Corneal Damage ................... 20
Conclusion .................................................................................................................... 33
References.................................................................................................................... 35
Abstract ......................................................................................................................... 38
1
Introduction
In the summer of 2003, the national media broke the story of Michael May, who,
though blinded as a young child, received a stem cell transplant that restored part of his
vision. At 3½ years old, May was blinded in a household chemical explosion. After some
time in the hospital, it was determined that he had only retained the ability to tell light
from darkness. He underwent three corneal transplants between the ages of 5 to 10 and
had a partial corneal graft at age 12, all of which failed due to severe scar tissue that
had formed from his injury as well as from each surgery. As a teenager, he lost the
ability to tell light from darkness in his left eye (May, n.d.).
May learned to live with his blindness and went on to lead a successful life. At
the age of 46, a doctor suggested that May might be eligible to undergo a new
procedure in which a stem cell graft would be used to replace the scar tissue and thus
allow for a corneal transplant. After several months of debating whether he wanted to
undergo another procedure, he agreed to have the stem cell transplant. His left eye was
too damaged to allow for any procedure, so only his right eye would receive the
transplant. In 2000, stem cells from an allogenic source were transplanted onto the
surface of his right eye. Later that year, he underwent a corneal transplant from another
donated cornea. He had a minor complication four months after surgery, when some
eyelashes irritated his new cornea, but the issue was resolved without further
complication. His stitches were removed, a rejection was reversed, and his eye was
deemed in good working condition (May, n.d.).
While May’s vision improved following the procedure, he still could not see
perfectly. He was able to catch a ball, recognize colors, weave through crowd without a
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cane or seeing-eye dog, discern two-dimensional objects, and recognize motion. He
was not able to recognize faces, including those of his wife and two sons, interpret facial
expressions, recognize stationary three-dimensional shapes; he also had a hard time
telling shadows from trees. He could not consistently distinguish a man from a woman
based on sight alone. To him a cube is a square with extra lines, until it is put in motion.
These problem areas remain four years after his operation (“Man’s Vision,” n.d.; Stein,
2003).
The outcome of May’s procedure has helped to clarify issues of the visual system
that were not understood before. His results suggest not only that we are born with
some hardwiring of vision that remains despite visual deprivation, but also that certain
aspects of vision develop at different stages in life. Some features are considered stable
in that once they develop, they do not change (such as motion detection), while other
features are plastic, requiring continual change and modification throughout life (such as
face and object recognition). If vision is lost before certain areas have had a chance to
develop, these areas may never be able to regain their function (“Man’s Vision,” n.d.).
May had brain scans done after the procedure to help clarify and support this
hypothesis. When he was shown a face or an object, the area of the brain that becomes
very active in a person with normal vision showed no activity at all in May. The motion
detection area of his cortex, however, appeared to be functioning normally when he was
shown an object in motion (Boyles, 2003; Stein, 2003).
Media reports of May’s procedure sparked interest about stem cell
transplantation in the lay public, but also piqued once again the curiosity of researchers
to discover more about the brain and its plasticity, as well as how to improve the
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procedure. The procedure has since been extended to include many other
injury/disease states resulting in corneal damage in hopes of restoring vision and
concretely answering questions surrounding visual development.
This literature review synthesized information about this new procedure and brain
plasticity, i.e., why did Michael May not regain full vision while many other who have
undergone this procedure have. Reports of trials and studies utilizing this procedure and
its variety of forms were reviewed. In support of this primary objective, the anatomy of
the visual system, visual development and processing, and the therapeutic use of stem
cells were also reviewed.
4
Stem Cell Theory
Stem cells are unspecialized cells that have the ability to regenerate themselves
via cell division for long periods of time. They are important to living organisms for
growth and development as well as regeneration. In the blastocyte, an embryo three to
five days old, approximately 30 stem cells give rise to the many highly specialized cells
that are needed to form a functioning adult. In the developing fetus, stem cells further
differentiate to become specialized tissue, such as the heart, lung, skin, etc. In adults,
stem cells are the replacement cells that replace damaged or dying cells after normal
wear and tear, injury, or disease states (National Institutes of Health [NIH], n.d.). The
ability of one cell to become any one of many possible cells makes stem cells
“pluripotent.”
All stem cells, regardless of location, have three unique features that distinguish
them from other cell types. First, they are capable of dividing and renewing themselves
through a process known as proliferation. A small starter population of stem cells will
proliferate for months, resulting in the formation of millions of cells. If all these cells are
the same as their parent stem cells, they are said to have carried out long term self
renewal. Second, they are unspecialized, which means that they have no specialized
function, other than to give rise to specialized cells. The third distinguishing feature of
stem cells, the process of an unspecialized cell giving rise to a specialized one, is
known as differentiation. Scientists believe that there are internal (genes) as well as
external (chemicals, physical contact with neighboring cells, molecules) signals that
trigger stem cell differentiation. The goal of some stem cell research today is to discover
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what these triggers are and use them to stimulate stem cells into specialized cells to
cure disease (NIH, n.d.).
Currently in the field of stem cell research there is controversy over where to
obtain stem cells: adult or embryonic sources. Embryonic stem cells are taken from
aborted fetuses and fetuses no longer needed from in vitro fertilization procedures. Less
controversial sources for embryonic stem cells are donated placentas or umbilical
cords. Adult stem cells are found in small numbers in many locations throughout the
body, including the brain, bone marrow, peripheral blood, blood vessels, skeletal
muscle, skin, liver, and the limbus of the eye (NIH, n.d.). They are thought to remain
within a specific area of tissue where they remain quiescent, that is, non-dividing, for
many years until they are activated by disease or injury and replace damaged or dying
cells by differentiating into the cells found in their present location. For example, stem
cells in the adult bone marrow give rise to the many types of blood cells (red blood cells,
white blood cells, and platelets). Under experimental conditions, however, these cells
can be induced to differentiate into cells other than cell types from their original location.
This phenomenon is known as plasticity of stem cells. The hope for stem cells is to
create new specialized cells with the hope of curing diseases, such as neurons to
produce dopamine (Parkinson’s disease), cells in heart muscle (damage due to
myocardial infarction), insulin producing cells of the pancreas (diabetes), and much
more including Alzheimer’s, spinal cord injury, stroke, burns, osteoarthritis, and
rheumatoid arthritis (NIH). This scholarly project focuses on the process of taking stem
cells from their present location, in this case the limbus, and using them for their original
function: to replace damaged or non-existing stem cells due to corneal damage.
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There are currently three methods that are used to try to identify adult stem cells.
First, cells can be labeled in a tissue with a molecular marker and observed for
differentiation. Second, the cells can be removed from a living tissue, labeled in a
culture plate, transplanted back into the tissue of origin, and observed for differentiation.
Finally, the cells can be isolated, grown in a culture plate, and manipulated to determine
what tissues they can become (NIH, n.d.).
The most widely known area of stem cell research utilized in the treatment of
disease is bone marrow transplantation. The stem cells in bone marrow can be used to
replace disease stem cells in patients with bone marrow disease of when a patient’s
bone marrow has been depleted due to treatment of disease (chemotherapy). The stem
cell research utilized for the treatment of corneal disease discuss in this project is very
similar. Patients with depleted limbal stem cells receive limbal stem cell transplants to
replace those lost to disease or injury states. While this utilization is still experimental, it
holds the promise to become another area of treatment in which stem cell
transplantation may become the gold standard.
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Anatomy and Physiology of the Visual System
In order to better understand limbal stem cell transplantation, the anatomy and
physiology of the visual system must be reviewed. The visual system consists of the
eye, cortical visual pathways, and cortical processing centers for vision. The purpose of
the visual system is to process the world around us and to make sense of it.
The Eyeball
The eyeball itself is the first structure to encounter the visual world. It measures
approximately one inch in diameter, with only the anterior one-sixth not encased by the
orbit of the skull. The eyeball consists of three layers: the fibrous tunic, the vascular
tunic, and the retina (Tortora & Grabowski, 2000).
The outer layer, the fibrous tunic, is made up of the cornea and the sclera. The
sclera, the white part of the eye, is made up of collagen and fibroblasts, and covers the
entire eyeball except the cornea and gives rigidity to the eye. The sclera meets the
cornea at a small opening known as the scleral venous sinus, or the canal of Schlemm
(Tortora & Grabowski, 2000).
The cornea is the transparent tissue that covers the iris. It serves two main
purposes in the visual system. First, it aids in protection for the rest of the eye, keeping
dust and germs from entering the center of the visual system. Second, the cornea acts
as the outermost lens for the visual pathway. It is curved allowing for bending and
focusing of light onto the retina. In fact, the cornea aids in focusing around seventy
percent of the light processed by the visual system (Eiad Eye Clinic, n.d.). The cornea
also serves as a filter for harmful light, filtering out most of the UV light from the sun.
8
The cornea is avascular, which means that it’s nutrients do not come from the
blood supply. It receives its nutrients from tears and the aqueous humor, fluid, located in
the chamber behind the cornea. The lack of blood vessels means that there is no
immune system protection against infection. However, if blood vessels were present,
even the smallest would take away from the cornea’s transparency, interfering with its
refractive purpose (Eiad Eye Clinic, n.d.).
Despite being only a few millimeters thick, the cornea has many layers within it.
Its outermost layer is the epithelial layer, the area of relevance to the discussion of this
review. It is this layer that the limbal stem cells travel onto to promote healing and
regeneration. The innermost layer of the cornea, the endothelial monolayer is a single
cell layer that separates the rest of the cornea from the aqueous humor of the eye. The
main function the endothelium is to pump excess water out of the stroma. Without this
single celled layer, the stroma would swell with excess water, and ultimately become
opaque, preventing proper refraction onto the retina. The stroma makes up
approximately 90% of the cornea. While the majority of this layer is water, it also
contains protein fibers that provide the cornea with additional strength and shape, as
well as cells that contribute in nourishment of the cornea. The arrangement and spacing
of the protein fibers are intricately designed to not interfere with the transparent nature
of the cornea (Eiad Eye Clinic, n.d.).
Descemets membrane is another thin, but strong layer that functions as a
protective barrier. This layer is also made up of collagen fibers. This layer readily
regenerates from the endothelial tissue that lies beneath this layer. Bowman’s layer is
the second outermost layer of the cornea. It is an acellular, transparent sheet of tissue.
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It is composed of collagen, which helps to provide the cornea with strength (Eiad Eye
Clinic, n.d.).
The epithelial layer, the outermost corneal layer, makes up about 10% of the
cornea’s thickness, and is approximately five cells deep (Eiad Eye Clinic, n.d.). This
layer of the cornea is composed of three sub-layers: the surface epithelium, the wing
cells, and the basal epithelium (Kaufman & Alm, 2003). The primary function of this
layer is protection of the eye from foreign materials. The smooth nature of the
epithelium allows for the absorption of oxygen. This layer also has thousands of nerve
endings, making it highly sensitive to any stimulus (Eiad Eye Clinic).
The cornea is an organ that continually regenerates itself in order to preserve its
function and to allow for rapid healing from minor insults. Mitosis occurs in the basal
epithelial cells. Cells from this layer of epithelium differentiate outward to form wing cells
and then surface epithelium, which degenerates and is sloughed off. This turnover
occurs in seven days. New basal cells originate from stem cells in the basal layer of the
limbal epithelium, a region of the eye peripheral to the cornea. The limbus contains
stem cells that differentiate into basal cells and then migrate onto the cornea. It is
important to note that mitosis stops when the cornea is wounded and resumes when the
wound is closed. This involves multiple chemical triggers outside the scope of this
review (Kaufman & Alm, 2003).
The vascular tunic or uvea of the eye has three parts: the choroid, the ciliary
body, and the iris. The choroid is highly vascular and lines the inside of the sclera. Due
to its high vascularity, it serves to provide nutrients to the retina. The ciliary body
extends from the anterior portion of the retina to the canal of Schlemm. The ciliary body
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has two functions: secretion of aqueous humor and alteration of the shape of the lens to
allow for near and far vision. The iris, the colored portion of the eye, is located between
the cornea and the lens and is attached laterally to the ciliary body. As a muscle, its
main function is to regulate the amount of light that enters the visual system through the
pupil, the hole in the center of the iris (Tortora & Grabowski, 2000).
The retina lines the majority of the posterior portion of the eyeball and is
considered the formal beginning of the visual pathway. It has two basic layers: the
pigment epithelium and the neural portion. The pigment epithelium is comprised of
melanin cells and lies between the choroid and the neural portion of the retina. The
melanin helps to capture stray light rays to prevent reflection and scattering of light
within the eyeball. Due to this process, the image cast on the retina by the cornea and
lens remains clear (Tortora & Grabowski, 2000).
The neural portion is considered an outgrowth of the brain and processes visual
information before transmitting nerve impulses to the brain. There are three distinct
layers within the neural portion of the retina: The photoreceptor layer (location of the
rods and cones), the bipolar cell layer, and the ganglion cell layer. These layers are
separated by two zones, the outer and inner synaptic layers, which is where synaptic
contacts are made between cells from the three layers of the retina. While the
photoreceptor layer is closest to the cornea, the path of light is actually in the opposite
direction. Light is reflected from the innermost layer, the layer closest to the retina,
outward to the photoreceptor layer, where the light is absorbed. The direction of visual
processing is then back toward the innermost ganglion cell layer of the retina (Tortora &
Grabowski, 2000).
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Visual Processing
Visual processing occurs in the following fashion. Light absorption takes place in
the photoreceptor layer by the rods and cones of the retina. The rods and cones then
send signals to the bipolar cells, making their synapses in the outer synaptic layer. The
bipolar cells then connect to ganglion cells via the inner synaptic layer. The ganglion
cells come together to form the optic nerve, which then transmits signals to the lateral
geniculate nucleus (LGN), to be discussed later (Daw, 1995).
There are two other cell types also located within the neural portion of the retina,
the horizontal and amacrine cells. These cells make lateral connections between cells
to further modify the visual information being sent to the LGN (Tortora & Grabowski,
2000). The horizontal cells make lateral connections between one photoreceptor cell
and another in the outer synaptic layer (Daw, 1995). The amacrine cells make lateral
connections between one bipolar cell and another in the inner synaptic layer. These
connections make modifications by comparing the light signals that fall on one section
of the retina with signals from another area (Daw).
Rods and cones are unique cells and aid in very different types of vision. Rods
have a “low light” threshold, which allows us to see in dimly lit environments. Because
the rods do not provide color vision, in dim light we only see shades of gray. Brighter
light activates the cones, which have a “high light” threshold and produce color vision
(Tortora & Grabowski, 2000).
There are three main processes involved in forming a clear image on the retina:
refraction of light by the cornea and lens, accommodation of the lens, and constriction of
the pupil. Refraction is the bending of light as it passes through material of different
12
densities. As light enters the eye, it is refracted by the cornea, and then again by the
lens to allow for immaculate focus onto the retina (Tortora & Grabowski, 2000).
The lens plays the main role in accommodation. When an object is curved, light
is refracted more from that object. The lens has the ability, with the aid of muscles, to
become more or less curved, depending on the nearness of the object being studied. As
an object moves closer, the lens becomes more curved via contraction of muscles and
light rays are refracted more, and the object remains clearly focused onto the retina
(Tortora & Grabowski, 2000).
Constriction of the pupil is made possible by the circular muscles located within
the iris. Constriction of the pupil prevents light from entering the eye through the
periphery of the lens. This in turn prevents a blurring of the image on the retina.
Constriction of the pupil occurs with accommodation as well as in bright sunlight to
preserve crystal clear focusing (Tortora & Grabowski, 2000).
The photoreceptors, rods and cones, together with photopigments play an
integral role in vision. Photopigments are proteins in the plasma membrane of the
photoreceptors. All photopigments contain two parts: a glycoprotein, opsin, and a
derivative of vitamin A called retinal. Retinal is the light absorbing portion of
photopigments. There is a different opsin for each different type of cone and another
type for rhodopsin. These types of opsins vary only slightly in the amino acid structure,
but these structural changes permit them to absorb different colors. Rhodopsin absorbs
blue to green light most effectively, while the three different cone photopigments absorb
blue, green, or yellow-orange light. When light rays enter the eye, they are absorbed by
the photopigments of the photoreceptors. Once they absorb light, photopigments
13
undergo a structural change that enables them to produce a receptor potential (Tortora
& Grabowski, 2000).
After the generation of the receptor potentials in rods and cones, these potentials
spread outward toward the synapses and cause the release of neurotransmitters by
rods and cones. These neurotransmitters induce potentials within bipolar and horizontal
cells. Bipolar cells are synapsed by 6-600 rods while cones synapse with just one
bipolar cell. Convergence by many rods onto a single bipolar cell increases light
sensitivity of rods, but tends to blur the image perceived. On the other hand, cone vision
is less sensitive to light, but has a higher acuity due to the one-to-one synapsing of
cells. Light stimulates rods to excite their bipolar cells, whereas light may excite or
inhibit bipolar cells of cones (Tortora & Grabowski, 2000).
Horizontal cells transmit inhibitory signals to bipolar cells lateral to the excited
rods and cones. This enhances contrast of the visual image. Horizontal cells also assist
in distinguishing colors. Bipolar cells excite amacrine cells, which synapse with ganglion
cells and transmit information to them. When excitatory signals are transmitted to the
ganglion cells, these cells become depolarized and initiate nerve impulses, which travel
through the optic nerve (Tortora & Grabowski, 2000).
The Visual Pathway
After the initial processing vision by the retina, the information is ready to be sent
to the brain. Ganglion cells from the retina join together to make the optic nerve, which
exits the retina and makes communication between the retina and the brain possible.
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The optic nerve passes through the optic chiasm, which is a crossing point for
fibers from the optic nerves of each eye. The fibers leaving the optic chiasm and
traveling to the LGN are known as the optic tracts and are therefore made up of axons
with nerve impulses from each eye. Fibers from the temporal half of each retina do not
cross over at the chiasm and continue on to the ipsilateral LGN, the LGN located in the
same hemisphere as the temporal retinal fibers, of the thalamus via the optic tract.
Nerve fibers from the nasal side of each retina do cross over at the optic chiasm and
travel to the contralateral LGN, the LGN in the opposite hemisphere of the nasal retinal
fibers. Branches from the axons of retinal ganglion cells also project to the midbrain to
help control pupil constriction (the pretectal area) and coordination of head and eye
movements (the superior colliculus). They also project to the suprachiasmatic nucleus
of the hypothalamus to help establish circadian rhythms. It is in the LGN that the fibers
synapse with neurons whose axons form the optic radiations that project to the visual
cortex on the same side (Tortora & Grabowski, 2000).
The lateral geniculate nucleus contains six layers of cells, with each layer only
receiving visual input from one eye. Optic tract fibers projecting from the ipsilateral eye
terminate in layers two, three, and five. Fibers from the contralateral eye travel to layers
one, four, and six of the LGN. Visual information is therefore still monocular in nature in
the LGN because no processing of the visual information from both eyes has occurred
(Gilman, 2003). The general function of the LGN is to channel visual information from
the retina to the visual cortex without much processing (Daw, 1995).
Fibers from the LGN travel in two pathways to reach the visual cortex, each
containing different aspects of the visual stimulus. Fibers from the lateral side of the
15
LGN travel down, forward, and then bend back, traveling through the temporal lobe and
then onto the occipital lobe. Fibers from the medial portion of the LGN travel somewhat
adjacent to the lateral pathway, but in a more direct route. These fibers keep their
topographic arrangement in order to present the information to the cortex the way the
retina saw it (Gilman & Newman, 2003).
There are two areas in the cortex that are directly related to vision. These are
called primary and secondary visual cortices. They are located in the occipital lobe of
the brain. The primary visual cortex receives information about the color, shape, and
movement of visual stimuli. This is the first area where information from both eyes
comes together for simultaneous processing. The visual association area (secondary
visual cortex) receives inputs from the primary visual cortex as well as from the LGN.
This area of cortex relates past and present visual information and is the area that
recognizes and evaluates what is seen (Tortora & Grabowski, 2000).
Both areas of the visual cortex are organized into vertical columns of neurons
that run from the surface into the white matter of the brain. These columns are called
ocular dominance columns and typically respond to information from just one eye. They
are very narrow and lie adjacent to one another. Cortical neurons respond to more than
just rays of light. The light that excites these neurons must have contrast and a specific
orientation within the visual field. This allows neurons across a series of ocular
dominance columns to respond to an ever-changing visual stimulus (Gilman &
Newman, 2003).
The cortex is also divided into six layers. Signals from the LGN come into layer
four which projects to neurons in layers two and three. These layers project to layer five
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which projects to the superior colliculus to further modify head and eye movements.
Layers two, three, and five also send further processed information back to the primary
visual cortex, which sends modified information back to the LGN (Daw, 1995). Working
together in this fashion, the visual cortices of the brain develop the complete picture of
the visual stimulus, thus allowing for binocular vision.
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Cortical Plasticity
During infancy and early childhood, important visual pathways are refined in the
brain. It is necessary that the layout and communication within the cortex be
changeable, plastic, with varying visual stimuli so that these pathways can be refined
and a few new ones be formed to accommodate for the vast variety of visual information
that is ever-changing. There are limited periods of time known as critical periods during
which the cortex is plastic. It is only during this time that important cortical changes can
be made. Once a critical period has passed, the cortical changes (or lack thereof) are
set. Changes that have not been made and even partial changes lead to full or partial
visual deficits.
For example, in normal development, modification of the cortex is necessary for
the development of binocular vision. Binocular vision develops when the input from both
eyes corresponds. During development, the increasing distance between the two eyes
throws off the correspondence of visual input. Because of critical periods and the plastic
nature of the cortex during this time, cortical neurons are able to adjust their
connections to both eyes. If the input from each eye is not able to correspond during
this time, binocular vision will not develop and the information from each eye remains
separate. Not surprisingly, the critical periods correlate with the time frame of the
greatest growth of the head and eyes (Kaufman & Alm, 2003).
In humans, critical periods for the visual cortex begin “between birth and six
months of age, peak from one to two years, and then begin to decline from three to
eight years” (Kaufman & Alm, 2003, p. 700), allowing the end stage fine tuning of the
visual system so that cortical plasticity is concluded by the age of eight (Daw, 1995). It
18
is known that depending on the area of cortex, critical periods vary. The critical period of
layer IV (the input layer from the LGN) ends sooner, making it stable earlier in
development than other layers of cortex (Daw). It is also known that some neurons have
greater plasticity than others depending on several factors (Kaufman).
Plasticity of the visual system is greater and lasts longer at higher levels of
processing. For instance, the retina is firmly established at birth, there is a small amount
of plasticity of the LGN, and the output layers of the visual cortex are more plastic than
the input layers. It is the output layers that project to the inferior temporal cortex that
recognizes faces and objects, information that changes and must be reinforced,
therefore requiring greater and longer lasting plasticity. The input layer (IV) processes
direction and orientation (Daw, 1995), which must develop early and must be grounded
in order for us to make sense of the world around us. There are areas of cortex that are
known to remain plastic in adulthood, the temporal cortex and the hippocampus. It is
only necessary for this discussion to know that visual memories are stored in the
temporal cortex, that the temporal cortex projects to the hippocampus, and that the
hippocampus is involved in generating our feelings toward situations.
As previously discussed, children’s brains are susceptible to visual changes until
the age of eight or nine years. Stimulus deprivation during these early years is likely the
most severe in terms of effects on the cortex. Short periods of stimulus deprivation
create more profound effects than do astigmatism and amisometropia (which can be
referred to as functional deprivations) present for the same time frame. In the case of a
congenital cataract, weeks of visual deprivation have effects on the cortex until 6-18
months of age, while months of deprivation can have effects until 8 years of age,
19
assuming the cataract is removed while the cortex is still plastic to all for some
development after removal (Daw, 1995). Corneal damage, like cataracts, is considered
to be a sensory deficit, or stimulus deprivation.
Michael May lost his vision at 3½ years old and didn’t regain vision until age 46,
well after the cortex had lost it plasticity. He was left with only cortical areas that had
passed their critical periods and were developed by the age of 3 years old, mainly the
input layer of cortex, which processes direction and movement. His biggest deficit is
facial recognition, which is related to the output layer of the cortex, which relies on a
lengthier critical period of plasticity for development, a period that he did not have due to
the chemical damage to his cornea.
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Stem Cell Transplantation to Reverse Blindness
Due to Corneal Damage
Stem cells have proven effective in the treatment of several diseases through
bone marrow transplantation. There is great hope for the treatment of many other
diseases as well. The newest organ that appears to benefit from the science of stem
cells is the eye. In recent years it has been discovered that when the eye is damaged
and vision is lost, the tissue around the cornea is no longer able to support the growth of
corneal tissue. Stem cells present in the limbus are concomitantly destroyed, so
transplantation of the cornea alone fails. Implanting stem cells along with a new cornea
has proven successful in maintaining the transplant as well as restoring vision.
Another recent addition to the procedure is the use of amniotic membrane as the
base for the new corneal transplant. Scheffer Tseng, a prominent researcher in the field,
states the “one thing we have known for decades is that if a wound occurs in the fetus,
after birth there is no scar” (Voelker, 1997, p. 1479). With this as his basis, he began
implanting amniotic membranes along with the new stem cell graft, in hopes that this
material would decrease the likelihood of scarring and further increase the success of
the procedure (Voelker). Another important feature of the amniotic membrane is that it
does not trigger an immune reaction in the recipient, making it one less aspect of the
procedure that needs to be typed to meet the immune status of the patient to prevent
rejection. Another interesting feature about the use of amniotic membrane is that this
substrate provides growth factors to the stem cells, further increasing the success of the
procedure (Ramaesh & Dhillon, 2003).
21
Bioengineered tissue has been widely used in the treatment of skin burns and
chronic skin wounds as well as cartilage replacement for knee injuries (“Eyesight
Restored”, 2000). It appears that limbal stem cell graft has been added to the list. The
general procedure is preformed by taking a biopsy portion of limbal tissue and placing it
onto a corneal graft bed in the lab. The stem cells grow and divide and form a one cell
thick layer (basement membrane). In the past, this layer, including remaining
undifferentiated stem cells, was then transferred to the damaged eye. Although there
were a few successful cases, the transfer process was extremely difficult and
researchers began looking for a more suitable “carrier” for the stem cells. In 1995,
researchers Kim and Tseng introduced the use of amniotic membranes to enhance the
limbal stem cell transplantation, as mentioned above. It did not take long for researches
in the ocular disease field to utilize this membrane for their procedures (Ramaesh &
Dhillon, 2003). Now limbal stem cells are removed and allowed to grow and divide on
this “transplanted basement membrane” (Ramaesh & Dhillon, p. 517), which facilitates
the growth of cells and allows the stem cells to more firmly adhere to the corneal graft
bed. This procedure is known as ex vivo (outside the body) cell expansion (Kim,
Djalilian, Schwartz, & Holland, 2003; Schwab, Reyes, & Isseroff, 2000) as well as being
described as an in vitro (outside the body and in an artificial environment) procedure
(Ramaesh). This form of bioengineered tissue, which mimics the natural corneal
surface, is then transplanted onto the patient’s defective eye(s), after the defect is
removed (“Eyesight Restored”, 2000; Schwab, et al.). There are many alternatives that
have been experimented with, while maintaining all of these basic components, such as
transplanting the amniotic membrane first and performing the limbal transplant at a later
22
date (Tseng, Prabhasawat, Barton, Gray, & Meller, 1998), rather than growing the stem
cells on the amniotic membrane and transplanting them as a unit (Sangwan,
Nemuganti, Iftekhar, Bansal, & Rao, 2003; Schwab, et al.; Tseng, et al.). The benefit of
doing the amniotic membrane transplantation before the allograft limbal transplant is to
reduce the inflammation to increase the success of the allograft limbal transplant
(Tseng, et al.). It appears that as long as the amniotic membrane and limbal tissue are
present and the entire defect is removed the procedure shows some level of success
when there is total limbal stem cells deficiency. When amniotic membrane is not
utilized, the success of the procedure is diminished (Pellegrini, et al., 1997).
Corneal Limbal Stem Cell Deficiency
There are many disease states that can cause corneal limbal stem cell deficiency
(LSCD), leading to the inability to maintain the cornea, resulting in corneal scarring and
loss of vision. Direct corneal damage can also cause direct damage to the limbus
causing LSCD. There are four main categories of conditions associated with LSCD.
Congenital conditions include aniridia, ectodermal dysplasia, and keratitis-ichthyosisdeafness syndrome. Traumatic conditions include chemical or thermal injury and
contact lens induced damage. Iatrogenic conditions can result from multiple limbal
surgeries and long-term topical medication use. Finally autoimmune conditions that can
cause LSCD are Stevens-Johnson syndrome (SJS), ocular cicatrical pemphigoid
(OCP), and atopic keratoconjunctivitis (Kim, et al., 2003; Schwab, et al., 2000; Tseng, et
al., 1998).
23
Typically what happens in these situations is the epithelial layer of the cornea
breaks down and conjunctival epithelium and blood vessels grow on to the cornea
(conjunctivalization). If LSCD is present, the cornea does not heal and scarring occurs.
Blood vessels from the conjunctiva remain causing corneal opacity (Kim, et al., 2003;
Pellegrini, et al., 1997; Tsai, Li, & Chen, 2000). This state often results in pain and
vision loss (Pellegrini, et al.).
As with any other treatment decision, several aspects of the disease or injury
must be taken into account to provide the best treatment option of the patient (amniotic
membrane transplantation alone, amniotic membrane transplantation with limbal
transplantation, or amniotic membrane transplantation with limbal transplantation with
follow-up keratoplasty to further reduce scarring) (Tseng, et al., 1998). Degree of LSCD,
severity of disease, and the presence of inflammation are the major three. Other
important features include unilateral vs. bilateral disease, adequacy of tear production,
presence of concomitant eyelid abnormalities, amount of scar tissue build-up, presence
of glaucoma, age of the patient, and the patient’s general health (Kim, et al., 2003).
Partial vs. total limbal stem cell deficiency is generally determined by the severity of the
disorder. Partial loss may be treated medically or with amniotic membrane
transplantation alone (Tseng, et al.), while extensive LSCD requires stem cell
transplantation to re-create a healthy ocular surface (Kim, et al.).
Scarring of the ocular surface, keratinization, is a risk factor for rejection and
should be removed prior to the transplant procedure (Holland, 1996). Tear film
contributes keeping the transplanted tissue moist and viable and promotes further
division of stem cells. Deficiency significantly contributes to failure of the transplant
24
(Shimazaki, Shimmura, Fujishima, & Tsubota, 2000). Inflammation of the ocular surface
should be treated before surgery with systemic immunosuppression (Kim, et al., 2003).
Patients whose disease state is characterized by chronic inflammation (SJS and OCP)
generally have a worse prognosis for this procedure (Kim, et al.; Pellegrini, et al., 1997;
Tseng, et al., 1998). Eyelid abnormalities may inhibit proper maturation of the transplant
by irritation. Glaucoma is also a risk factor for failure and is commonly seen in patients
with LSCD. In patients with uncontrolled glaucoma, or those who require multiple
medications to control their intraocular pressures, a tube shunt procedure should be
considered before transplantation, due to the fact that glaucoma medications can be
toxic to the new transplant. Systemic immunosuppression while vital to the success of
this procedure may not be tolerated by geriatric patients or patients with concomitant,
chronic, long-term disease. Younger patients also present a problem, as they are harder
to examine due to lack of cooperation. Anesthesia may be required at each postoperative visit to properly examine the new transplant (Kim, et al.).
Sources for Limbal Stem Cells
Limbal tissue is taken from different sources depending on unilateral or bilateral
damage in the patient. If the damage is unilateral the patient may donate limbal tissue
from his remaining healthy eye (autologous transplantation). However if the damage is
bilateral, the patient must rely on allogenic sources, i.e. donor limbal tissue that has
been HLA screened and is compatible with the patient’s HLA status in order to prevent
rejection. The typical sources for this type of transplantation are from living related
donation or cadaveric donation (Kim, et al., 2003).
25
Tseng first published results on autologous transplantation. The concern at this
early phase in the technology was inducing LSCD in the healthy eye of the patient. He
came to the conclusion that the risk is relatively low as long as the eye is truly healthy
and only the minimal amount of limbal tissue is taken (Kenyon& Tseng, 1989). The
greatest benefit of this procedure is the elimination of graft rejection and no need to use
systemic immunosuppression (Kim, et al., 2003; Pellegrini, et al., 1997; Sangwan, et al.,
2003; Tsai, et al., 2000; Tseng, et al., 1998).
Living related conjunctival limbal allograft is one of the possibilities for bilateral
disease. This procedure utilizes the limbal stem cells from a living relative rather than
taking them from the patient’s own eye. Like any organ donation, it is important to
properly HLA type all potential donors. The presence of glaucoma or history of contact
lens wear excludes someone from being a donor. Screening for HIV and hepatitis B and
C should also be preformed. This procedure provides some degree of histocompatibility
due to HLA matching, but systemic immunosuppression is still required to further
decrease the risk of rejection (Kim, et al., 2003).
Keratolimbal allografts are the most prominent source of limbal stem cells; it
utilizes cadaveric stem cells. This technique has seen the greatest evolution. In 1984,
only the cornea from the cadaver was used, but success was limited due to the lack of
limbal stem cell use (Thoft, 1984). He later added the use of limbal stem cells to his
technique in 1990 (Kim, et al., 2003). After several more years of experimenting, the
current technique of using stem cells alone grown of amniotic membrane was utilized
(Sangwan, et al., 2003; Schwab, et al., 2000; Tseng, et al., 1998). Keratolimbal
allografting alone is useful when only LSCD is present in bilateral or unilateral disease;
26
however, when significant scarring or damage to the surrounding conjunctiva is present,
additional procedures (corneal and/or conjunctival cell transplantation) are necessary
(Kim, et al.).
Stevens-Johnson syndrome, OCP, and chemical injuries are usually the more
severe forms of corneal disease and usually cause total LSCD. Stevens-Johnson
syndrome and OCP often are accompanied by inadequate tear production due to the
amount of chronic inflammation associated with these disease states. As stated above,
inadequate tearing makes it difficult for the transplant to survive and can be corrected
with punctal duct occlusion (Kim, et al., 2003). In fact, the ocular surface should be as
near to normal as possible to increase the success of the procedure (Ramaesh &
Dhillon, 2003).
In the past, due to inadequate stem cell harvesting, the proper numbers of stem
cells were not obtained from donors. This led to gaps in the recipient’s new transplant.
These areas made prime targets for conjunctivalization. In the current combined
procedure, limbal tissue is obtained from a compatible living related or cadaveric donor
and is placed on storage media in the lab. Corneal graft tissue is placed superiorly and
inferiorly on the prepared ocular surface while keratolimbal tissue fills in nasal and
temporal zones. Systemic immunosuppression is required; these patients are at a
higher risk for rejection because two different types of tissue are used (Kim, et al.,
2003).
27
Limbal Stem Cell Harvest and Graft Preparation
It is believed that a limbal biopsy approximately 2mm2 holds a sufficient number
of stem cells for the procedure. Once the biopsy of limbal tissue is in the lab it is washed
in phosphate buffered saline. The biopsy is then cut up and dissociated with
trypsin/ethylenediaminetetraacetic acid. The matter is then placed in the centrifuge and
the aggregate removed. This single suspension is cultivated as described above on an
amniotic membrane substrate (Ramaesh & Dhillon, 2003; Schwab, et al., 2000).
Amniotic membrane used for the procedures generally has a stem cell density of 1.5-3 x
106 cells/cm2 (Schwab), or a sheet approximately 4x3cm (Sangwan, et al., 2003; Tseng,
et al., 1998). In order to be used for the expansion of stem cells, all of the amniotic
epithelial cells must be removed. This is accomplished by a combination of trypsin and
mechanical scraping (Koizumi, Inatomi, Suzuki, Sotozono, & Kinoshita, 2001; Ramaesh
& Dhillon; Schwab, et al.). The prepared limbal stem cells are typically cultured on the
amniotic membrane for two to three weeks (Tsai, Li, & Chen 2000).
Post-Operative Patient Management
Systemic immunosuppression recommendations include a multidrug regimen.
The three drugs most commonly used are prednisone, cyclosporine or tacrolimus, and
azathioprine or mycophenolate mofetil. These medications are generally continued 1218 months at which point tapering of the dose is considered (Kim, et al., 2003).
Postoperative management includes use of topical corticosteroids and topical
cyclosporine to reduce inflammation. Topical antibiotics have also been used to prevent
infection (Tseng, et al., 1998), and in some studies oral antibiotics have been used
28
(Sangwan, et al., 2003). As stated above, tear production is vital to the survival of the
transplant. Punctal duct occlusion is preformed when deemed necessary, and artificial
tear use is generally used in all cases (Kim, et al., 2003; Sangwan, et al.; Tseng, et al.).
If there is a delay in the formation of the corneal epithelium (epithelialization)
tarsorrhaphy is preformed. In this procedure the eyelids are entirely or partially sutured
together to allow epithelialization to occur in a protected environment (Kim, et al.).
Results from Clinical Studies
Allogenic Donation
In a retrospective study reported in the journal Ophthalmology, thirteen eyes from
eleven patients were evaluated after having undergone stem cell transplantation. All
eleven patients were diagnosed with total limbal stem cell deficiency. Five eyes suffered
from acute SJS and two with chronic SJS, one had recently been damaged in a
chemical injury and two had chronic chemical injuries, two eyes had OCP, and one eye
had drug-induced damage. In this study, the corneal limbal epithelium was allowed to
culture for four weeks on an amniotic membrane carrier. Before implantation, the
cultures showed four to five layers of well-differentiated cells. Systemic
immunosuppression was administered before the procedure and some amount of
immunosuppression was continued for six months after the procedure. Patient progress
was monitored on a regular basis. Forty-eight hours after the procedure all 13 eyes
were noted to be clear and smooth and the same results were found five days after the
procedure, suggesting that the transplantation was complete and holding. In all cases,
the conjunctival inflammation due to surgery subsided. In this study three eyes (two
29
patients) rejected the transplant. These were patients who had suffered from an acute
chemical burns and acute SJS. These patients were 21 and 32 years old. A third
patient, seven years old, showed some signs of conjunctival epithelialization, but did not
suffer visual impairment due to this process. This patient also suffered from bilateral
acute SJS. All 13 eyes of all 11 patients experienced some level of visual improvement
after the procedure and no patient had recurrent neovascularization at the end of the
follow-up period. Patients with OCP showed the least amount of visual improvement,
likely due to chronic inflammation associated with the disease (Koizumi, et al, 2001).
A second study reported in the 2003 Journal of the American Academy of
Ophthalmology, reported on the allogenic stem cell transplantation in one 50-year-old
female patient who suffered from bilateral alkali chemical burns. After her injury, she
was count-fingers at three feet in her right eye and count-fingers at four feet in her left
eye. Eight months after her injury, she underwent cadaveric limbal stem cell
transplantation with amniotic membrane as a carrier. Proper immunosuppression was
administered before, during, and after the procedure. She was routinely monitored after
the procedure, and at the 6-month post-operative visit, her visual acuity had improved to
20/100 bilaterally (Espana, Grueterich, Ti, & Tseng).
Autologous Donation
In a study reported in a 2000 issue of the New England Journal of Medicine, six
patients with unilateral corneal disease underwent autologous stem cell transplantation
with amniotic membrane as a carrier. The study group suffered from partial and total
limbal stem cell deficiency. Three patients suffered from chemical burns, two from
30
pterygium, and one patient with chronic inflammation due to disease state.
Immunosuppression was not necessary in this study due to autologous donation. After
the procedure, all transplants grew and held. Five of the six patients showed visual
improvement to some degree, with the greatest improvement seen in the patient with
chronic inflammation (20/200 to 20/50 visual acuity). The patient who did not improve in
visual acuity maintained his 20/20 vision. All six patients were followed after the
procedure for approximately 15 months, during which time no patient exhibited
neovascularization or inflammation (Tsai, et al.).
In a study reported in Lancet, two patients with severe unilateral alkali burns
underwent autologous stem cell transplantation. One patient had suffered the burn 22
years prior to the procedure and the other 10 years prior. Amniotic membrane was not
used and instead, anti-inflammatory drops were placed into the surgical eye twice a day
after the procedure for 10 days, after which time the patients continued to used artificial
tears four times a day with an anti-inflammatory drop twice a day. Both patients
benefited from this procedure from a comfort aspect; pain was decreased in both
patients. However, only mild visual improvement was reported in this study (Pellegrini,
et al., 1997). This could be due to the inflammation that recurred as a result of not using
amniotic membrane as the carrier, as well as inflammation due to the several other
procedures each patient had undergone. Also, the limbal biopsy was only 1mm2, so it is
also likely that insufficient stem cell numbers were obtained.
A third study was reported in the journal Cornea in 2003, in which a 31-year-old
female patient who had suffered from chemical burns underwent autologous donation
and transplantation. The uniqueness of this study is that she suffered from bilateral
31
disease, but only one eye had total limbal stem cell deficiency. The biopsy was taken
from the eye that was partially deficient, and with extra time, grew enough stem cells to
allow for two sheets of transplant material with amniotic membrane as a carrier for both
sheets. Donation was taken from the left eye; visual acuity in this eye was count-fingers
at three feet. Her right eye had a visual acuity of 20/400 pre-operatively. At her one-year
post-operative visit there was cosmetic improvement as well as improved visual acuity
in the right eye to 20/40 with the acuity in the left eye remaining count-fingers at three
feet (Sangwan, et al.). Speculation suggests that the biopsy might have been too large
from the partially deficient eye or that the damage in the left eye was actually more
significant than initially thought.
Use of Amniotic Membrane
In 1998, in a study reported in the Archives of Ophthalmology, researchers
studied the effectiveness of the use of amniotic membrane for the procedure of limbal
stem cell transplantation. The researchers studied 26 patients (31 diseased eyes) with
varying degrees of limbal stem cell deficiency. Fourteen eyes suffered from chemical
burns, five eyes from SJS, three eyes with contact lens induced disease, three eyes
with aniridia, two eyes with iatrogenic stem cell deficiency, two eyes with atopy, and two
eyes with an unknown cause for limbal stem cell deficiency. The researchers divided
the eyes into three groups: group A (mild disease, 10 eyes), group B (moderate
disease, seven eyes), and group C (severe disease, 14 eyes). Group A received
amniotic membrane transplantation (AMT) alone, group B received AMT and allograft
limbal transplantation (ALT), and group C received AMT, ALT, and follow up
32
keratoplasty to further reduce scarring. All patients except those in group A received
immunosuppressive treatment. All eyes, except the two with atopy, showed
epithelialization in two to four weeks and decreased inflammation, scarring, and
vascularization. All of the surfaces were smooth. Visual acuity was improved in 25 of the
eyes, with decreasing visual acuity improvement with increasing severity of disease. All
eight eyes in group A showed visual improvement, five of seven eyes in group B
showed improvement, and 11 of the 14 eyes in group C showed some visual
improvement. Nine eyes in group C eventually showed signs of rejection, and three of
the remaining 21 eyes in groups B and C showed early, yet reversible signs of rejection.
The researchers came to the following conclusions. For patients with partial limbal stem
cell deficiency with only superficial involvement of the cornea, AMT alone can be utilized
and is sufficient. They considered this method to be superior to the combination with
ALT because no immunosuppression was required. However, when there is total limbal
stem cell deficiency ALT is also needed to replace the limbal stem cells. They
concluded that the decrease in inflammation and vascularization that is provided with
the use of AMT enhances the success of allograft limbal transplantation (Tseng, et al.)
33
Conclusion
Stem cell research is an ever growing and always fascinating area of science. It
has been helping patients with a variety of diseases through bone marrow
transplantation. Many other diseases are on the horizon of being cured with stem cells.
The closest disease state to reaching the “cure status” with the aid of stem cells is
ocular surface disease due to corneal damage.
As clinicians, it is important for physician assistants to be well read in the more
advanced procedures that patients may commonly ask about. While limbal stem cell
transplantation is still in the experimental stage, this procedure shows great promise
and will likely overcome the current barriers that are holding it back. Physician
assistants should be prepared to recommend this procedure in the future and refer to
the appropriate specialists when the time comes.
There are several areas surrounding this procedure that need improvement
and/or further research. First, while researchers think that only a 2mm2 biopsy holds a
sufficient number of stem cell for a successful outcome, it is possible that either a larger
number is initially needed or that more time is needed to allow a greater number of cells
to grow and divide for a higher success rate.
Secondly, while previously proposed methods can determine whether a cell is a
stem cell, this is a long process. Research is currently focusing on identifying a “stem
cell marker” which would accurately and quickly tell researchers if a cell is a stem cell.
Researchers think that a gene p63 (a variant of p53) is an accurate limbal stem cell
marker, but further research is necessary (Ramaesh & Dhillon, 2003).
34
Third, while the amniotic membrane is a viable substrate for stem cells to grow
and be transplanted on, the procedure might show greater success if an actual
bioengineered cornea were created. Amniotic membranes would likely still be used as
the carrier for limbal stem cells, but the corneal transplant portion of the procedure
might show greater success with a bioengineered tissue.
Fourth, long-term success has not yet been shown since the procedure is still
relatively new. It is unknown how long the current transplants will last. What is the longterm viability of the amniotic membrane as a carrier for the stem cells? Once the stem
cells are transplanted, will they truly maintain the features that identify them as stem
cells, mainly proliferation? These are questions that remain to be answered.
Finally, as with any organ or tissue transplant, factors associated with transplant
rejection need to be discovered. What causes spontaneous rejection in a seemingly
successful transplant? Infection? Improper immunosuppression? Further investigation
needs to be conducted.
Michael May was a unique patient. In all studies reviewed, no other patient had
been injured at such an early age and received the procedure after such an extended
period of time. As a result, he is the only patient to have had results that have shed light
on the cortical plasticity issues discussed in this project. Because of Michael May’s
outcome we know that there are critical periods in visual development as well as visual
characteristics that develop at different times to allow for the growth of the head.
Stem cell research is a rapidly growing field. Limbal stem cells are the most
recent success story in the saga. It is only a matter of time before stem cells are used to
cure diseases such as Parkinson’s disease, diabetes, and Alzheimer’s disease.
35
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38
Abstract
Objective. This project reviewed the current status of limbal stem cell transplantation to
reverse vision loss due to corneal damage. Method. Relevant articles from searches of
PubMed were used to compile the review. Discussion. Studies have looked at
autologous and allogenic donation of limbal stem cells using a donated amniotic
membrane as a carrier base to reduce scarring. This technique has shown a decrease
in scarring, replenishment of limbal stem cells, and correction of vision to varying
degrees in the majority of patients studied. In addition, the restoration of partial vision in
one patient who was blinded as a young child provided anecdotal evidence for the
critical periods of cortical plasticity; certain aspects of his vision did not return because
they never developed. Conclusion. Limbal stem cell transplantation is showing
promise as a treatment for corneal damage. It also provides an opportunity to learn
about the development of the visual cortex.