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- I would like to thank the organizers
for giving me the opportunity to share our work,
to develop this Limbal Stem Cell Therapy.
My presentation will be divided into four parts.
Background then development of therapeutic candidate
followed by development of the outcome measure,
and finally, the Phase 1 clinical trial design.
Cornea is the transparent part of the eye,
is lying behind epithelium
and this epithelium is maintained by a group
of epithelial stem cells that reside in the limbus,
which is junction between the cornea and conjunctiva.
And the basal layer of the limbal epithelium
and the also layer harbors this group of stem cells.
When they are deficient or lack of function of stem cells
that lead to a disease entity
called Limbal Stem Cell Deficiency.
The presentation of this disease varies based on the degree
of the Limbal Stem Cell Deficiency.
In partial disease, only a sector
of the cornea and limbus is affected,
in total Limbal Stem Cell Deficiency,
the entire cornea is affected.
Cornea transplant does not treat Limbo Stem Cell Deficiency.
In eyes with normal stem cell function,
the graph can remain clear and survive for a longer time,
but in eyes with limbal Stem Deficiency,
the graph usually fails within a couple of years.
The cause of Limbal Stem Cell Deficiency could be due
to genetic disorders or due to injury.
And any injuries to the limbus can lead
to stem cell deficiency, which include chemical injury,
contact lens wear or chronic inflammation.
It's not until recently that the first global consensus
on this disease entity was developed.
There are four take home messages.
The first is that the Homer of the disease
is the cornea epithelium is replaced
by the conjunctiva epithelium.
The second is that a diagnostic test is recommended
in addition to clinical exam.
Third autologous stem cell therapy is preferred
over allogeneic therapy.
And lastly, limbal stem cell treatment is preferred
over keratoprosthesis, which is the artificial cornea.
We also conducted a Meta-analysis
on the outcomes of a Limbal Stem Cell Transplant.
We found that autologous Limbal Stem Cell Transplant
had a higher success rate and lower complication rate
than did allogeneic transplant.
And the long term success rate is below 76%.
The criteria of diagnosing
and staging Limbal Stem Cell Deficiency
and the outcome measure of any treatment
vary greatly among all studies.
And we concluded that randomized control clinical trials
are necessary to compare the efficacy
among different therapies.
The first approved Stem Cell Therapy
was first reported back in 1997.
In this first trial of therapy,
a small biopsy was taken from the donor eye,
which is the healthy eye and these stem cells,
where then expanded in the dish,
and then transplanted onto the other eye,
which is disease eye,
and this ocular surface was restored.
The therapy was approved
by the European medicine agency called Holoclar.
There are several disadvantages of this therapy.
First, it requires animal products.
Second, it treats mostly unilateral disease
and it lacks a standardized outcome measure.
And the success rate again was limited to 76%
with repeated treatment.
So looking forward, we are seeing that the therapy
should be safe, which means that
it should eliminate animal products
to avoid cross-contamination.
It should be efficient,
which means that the expansion of the stem cell population
in culture should allow for a higher success rate.
And third, it should be very effective.
That means a higher long term survival
of these transplant cells.
So the next session we want to share with you
how we develop a new therapy using
cultivated autologous limbal stem cells.
We focus on the manufacturing process
of this autologous cultivated limbal cells.
We want it to be feasible, efficient
and GMP-compliant, and Xeno-free.
To achieve that we optimize the culturing process
by improve in the following categories.
First is the starting material,
which means that when the tissue is biopsy,
how to be culture the stem cells from the biopsy.
Secondly, are they supporting cells/feeder cells if any,
that will increase the efficiency of the expansion
of the stem cell population.
And third, how can we ensure that culture substrate
or the transplant substrate safe and effective,
and the next, the culture media,
how can we make it a xenobiotic-free?
And lastly is the way that the cells are culture
and how can we recreate the in vivo limbal stem cell niche.
So after comparing more than 70 different culture methods,
and you finally develop a Xenobiotic-free
and feeder-free culture system
by preserving the native stem cell niche driven culture,
and then replacing the fetal bovine serum with human serum,
and also a removal of the DMSO
and cholera toxin during this process.
Next, to compliant with the directory requirement,
we also develop IPCs in process controls
and also released criteria so that we ensure the quality
of the cultivated limbal stem cells.
Next I'm ready to talk about the development
of standardized clinical outcome measures.
Clinical presentations might not reflect the actual
stem cell function and a quantitative staging system
is still lacking and needs to be developed to quantify
in vivo limbal stem cell function and outcomes
of the stem cell treatment.
We utilize a very powerful in vivo imaging,
In Vivo Confocal Microscopy,
a hand image, all layers of the cornea,
different layers of the epithelium and the junction
of the conjunctiva and cornea, which is the limbus.
You first set out to looking to the micro structural changes
of the epithelium by imaging the central cornea
and four regions of the limbus.
Even in earlier stage in the epithelial cells become larger
and the nuclei become more prominent.
This changes we call it metaplastic change
even become more apparent in the intermediate stage
and the cells with normal looking cornea phenotype
missing in the eyes with late stage.
A similar picture is seen in the limbal regions.
Often we see an influx of inflammatory cells.
At the same time,
we also develop a quantitative clinical scoring system
that takes into consideration of the extent
of the limbal and corneal involvement,
as well as the central involvement.
And each eye will be giving a number to quantify
the degree of the clinical degree.
We're looking into 24 in vivo parameters of eyes
with Limbal Stem Cell Deficiency, basal cell density,
epithelial thickness in five locations of ocular surface,
central cornea, and four regions of the limbus.
Cell morphology, central cornea subbasai nerve density
and nerve branch scores.
The basal cell density decrease in the eyes
with Limbal Stems Deficiency in the both the central cornea
and the limbus.
The degrees of decrease correlate with the degree
of the severity of the disease.
Epithelial thinning also was detected in eyes
with that stem cell deficiency.
In normal eye the epithelium is visualized
by another, in vivo imaging modality, Anterior segment OCT,
Optical Coherence Tomography in eyes
with stem cell deficiency
the layer of this epithelium becomes thinner
and often absent in advanced stage
of limbal stem cell deficiency.
And the decline of Subbasal Nerve Density
was also observed again,
the degree of decline has positive correlation
with the degree of the deficiency.
A Molecular Diagnostic test also is essential
to confirm the diagnosis of Limbal Stem Cell deficiency.
We found cytokeratin 13 is a specific marker
of the conjunctival epithelium
by sampling the ocular surface on the cornea,
on the conjunctiva and the cells
can be subject to immunohistochemistry staining
and case study in positive cells on the cornea
signify Limbal Stem Cell Deficiency.
So based on our findings, we include;
clinical scores, basal cell density, subbasal nerve density,
epithelial thickness of the central cornea
and molecular diagnostic tests using keratin 13 as a marker
could be used to diagnose
and stage Limbal Stem Cell Deficiency.
And this criteria will be used as outcome measure
of our ongoing limbal stem cell trial.
In this last session,
I will introduce you to our phase 1 clinical trial.
The purpose of this clinical study is to investigate
the safety and feasibility,
hopefully part of the also efficacy
of our cultivated autologous limbal stem cells
for the treatment of Limbal Stem Cell Deficiency.
We also want to validate the Limbal Stem Cell Deficiency,
diagnostic criteria and quantitative seizing system.
This is a randomized control phase 1 clinical trial
and a single center.
20 subjects will be recruited.
The first five will undergo limbal stem cell treatment,
and the subsequent 15 will be randomized in a 2:1 ratio
into the stem cell treatment
or the scleral lens treatment group.
This subject will be undergo screening
using the clinical grading system,
the in vivo imaging and molecular diagnostic test
to determine the degree
of their Limbal Stem Cell Deficiency.
If they meet the inclusion criteria,
the biopsy will be taken from the donor eye,
grow them in culture and then transplant it back
to the other eye, the disease eye.
The control will be a scleral lens human group.
You can contact our study coordinator through email
or to me, if you have any additional information
and then could try register at the clinicaltrials.gov.
I would like to thank my teams who have contributed
to the work that I have presented.
Sheyla Gonzalez is the project manager who developed
the Xeno-free and feeder-free culturing system.
Ellen Pascual is our clinical research center administrator
who helped us tremendously in the entire project.
Alpha Stem Cell Clinic and this Stem Cell Center UCLA
have assisted us on the ID preparation.
Dr. Donald Kohn is the director of the GMP facility,
and he has helped us greatly on the GMP tech transfer
and also the GMP manufacturing all our product.
The portal has been funded from the infancy
by the California Stem Cell Agency,
as well as the National Eye Institute.
And the other funding agency also contribute
to the beginning of the project.
Thank very much for your attention.
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