Guidance on Evaluation of Autologous Induced Pluripotent StemCells-derived Retinal Pigment Epithelial Cells

1. Introduction
The fundamental technical requirements of the quality and safety of the medical devices or
pharmaceuticals processed from autologous human induced pluripotent stem (iPS) cells or the
iPS-like cells are stipulated in the Notification Pharmaceutical and Food Safety Bureau (PFSB)
0907 No. 4, “Guideline on ensuring the quality and safety of human autologous induced
Pluripotent Stem (-like) cells-derived medical devices or pharmaceuticals” issued by the
Director of the PFSB of the Ministry of Health, Labour and Welfare (MHLW) on September 7,
2012.
This guidance shows specific points to consider in the evaluation of quality, efficacy and safetyof the autologous human iPS (or iPS-like) cells-derived medical devices that can be speciallyintended to be used in the treatment of retinal pigment epithelial disorders in addition to thefundamental technical requirements in the Notification mentioned above.
2. Scope
This guidance identifies points to consider in the evaluation of the quality, efficacy, and safetyof medical devices processed from autologous human iPS cells, specifically intended to be usedin the treatment of retinal pigment epithelial disorders in addition to the fundamental technicalrequirements.
Further, when it is difficult to determine whether the product being developed could be
categorized as a medical device, please consult with the Office of Medical Device Evaluation of
the PFSB, the MHLW, if necessary.
3. Role of this guidance
In consideration of the fact that the scope is medical devices processed from autologous
human iPS cells that are being developed based on the remarkable technological advances inthis area, this guidance is designed to indicate current issues instead of providing acomprehensive list of problems and points to consider. Therefore, it will be revised astechnological innovation progresses and knowledge accumulates, and it does not mandate what
information must be included in application document for approval.
For the evaluation of products, it is necessary to have a thorough understanding of thecharacteristics of individual products and to handle the data in a flexible and scientificallyrational manner.
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It is also recommended that other relevant guidelines from both Japan and overseas should be
consulted in addition to this guidance.
4. Terms and definitions
For the purposes of this guidance, the definitions given in the Notification PFSB 0907 No. 4
above and the following apply.
(1) Retinal pigment epithelial cells: The outermost layer of the 10 retinal layers. These cells
form a single layer of epithelial cells and have the ability to phagocytose retinal
photoreceptor cells and regenerate visual substances (such as retinal), forming theblood–retinal barrier. These cells are the main sites of lesions in age-related maculardegeneration.
(2) Retinal photoreceptor cells: One of the types of cells in the retina. They are called “light
receptors,” converting light energy into electrical energy. They are located at theoutermost layer of the neural retina, and their apical regions called “outer segment” isregularly phagocytosed by the retinal pigment epithelia and replaced by new discmembranes that form those outer segments.
(3) Phagocytic ability: Similar to macrophages, retinal pigment epithelial cells have the ability
to incorporate and digest foreign objects, such as bacteria and cell debris. Under normal
circumstances, they regularly ingest the apical region of photoreceptor cells.
(4) Barrier function: Retinal pigment epithelial cells are bound to each other by an adhesive
structure that is impervious to substances. This feature is known as the barrier function.
(5) Cell sheets: This refers to cells that gather to form a sheet-like structure.
(6) Subretinal transplantation: This refers to a surgical treatment consisting of deliberately
creating a space in the subretinal region, which is located between the sensory retina andretinal pigment epithelial cells, in which tissues or devices are inserted.
(7) Exudative lesions: This refers to a pathological condition in which choroidal
neovascularization occurs in association with age-related macular degeneration. Causedby disturbances in the structure of the retina due to the accumulation of exudates andneovascular tissues beneath the retina, the condition involves rapid and severe decrease invisual acuity.
(8) Examination of the ocular fundus: This refers to a test aimed to identify changes in the retina
and choroid by using indirect ophthalmoscope, direct ophthalmoscope, front lenses, etc,
projecting light into the ocular fundus from the front of the eye, through the pupil.
(9) Fundus angiographic test: In this test, a fluorescent substance (fluorescein) is administeredintravenously, and the ocular fundus is examined and photographed using a fluorescentcamera. This test assists in the evaluation of the barrier function and hemodynamics of the2

ocular fundus, as well as in the detection of neovascularization.
(10) Retinal tomography: This is also known as optical coherence tomography (OCT) and is atest that allows the cross-sectional observation of the layers of the retina in vivo. It iseffectively used in the detection of choroidal neovascularization and retinal detachment.
(11) Retinal sensitivity test: This test examines the area of the subject’s visual perception by
projecting small lights on the retina and by changing the brightness of each light. It
includes tests such as microperimetry and static quantitative perimetry.
5. Points to consider in evaluation(1) Quality control of products[1] Characterization items on specification of quality as retinal pigment epithelial cellsa) Cell morphology
Phase contrast microscopy allows visual examination of the specific morphology toretinal pigment epithelial cells (e.g., brown pigments and polygonal, pavement-likeshape).
b) Genes specifically expressed in the retinal pigment epithelial cells
Confirm whether retinal pigment epithelium-related genes (such as RPE65, CRALBP,MERTK, and BEST1) are expressed.
c) Cell purity
Cell purity may be confirmed by immunostaining of the combination of more than
one antibody such as against RPE65, bestrophin, and PAX6. Alternatively, it may be
confirmed through the objective quantification of the number of pigmented cells by
microscope-image analysis. Because almost all the pigmented cells in a characteristic
morphology from purified cultures of cells with relevant genes are judged as retinal
pigment epithelial cells.
d) No undifferentiated cells in the productFlow cytometry by using immunostains for undifferentiated-cell markers (i.e., Oct3/4,
Sox2, and TRA-1-60) and quantification of marker genes (e.g., OCT3/4, Nanog, andLin28) through quantitative reverse transcription-PCR have been shown in the literatureas the methods to detect residual undifferentiated cells in the product.
Among them, the investigation of the Lin28 expression by quantitative RT-PCR ishighly specific and sensitive to find the undifferentiated cells1), and thus, can be a
representative method in the evaluation.
In addition, the residual of undifferentiated iPS cells in the product should notnecessarily be considered as a sign of tumorigenicity, and items used in non-clinicalstudies should be referred for tumorigenicity tests.
3e) Functional evaluation
The functional properties of retinal pigment epithelial cells compatible for therapeutic
use should be checked during the manufacturing process. The following are examplesof the commonly performed tests.
・Phagocytic ability: Incorporation of fluorescence-labeled outer segments of theretinal photoreceptor cells or the incorporation of fluorescent beads

added to theculture medium into cells is evaluated using flow cytometry.
・Growth factor secreting ability: The secretion amount of vascular endothelial growth
factor (VEGF), pigment epithelium-derived factor, and so on are measured using
enzyme-linked immunosorbent assay.
[2] Characterization items on specification of quality as retinal pigment epithelial cell sheets
When analyzing the characteristics of retinal pigment epithelial cell sheets, the validity of
preparation process of cell sheets in manufacture is clarified by confirmation of themorphology and evaluation of mechanical compatibility and functional properties as shown
below.
a) To confirm sheet morphology, tissue sections of the sheet are examined, or
three-dimensional observation of the sheet by using a confocal microscope isconducted to confirm that cells form a sheet.
b) To check the mechanical compatibility, a graft prepared from the cell sheet detached isverified the absence of breakage or damage in the sheet.
c) To examine the presence of functional properties (barrier function), the expression ofmarkers associated with barrier function is observed by immunostaining (ZO-1
staining). Or transepithelial electrical resistance and such are measured.
(2) Non-clinical tests[1] Tumorigenicity tests
To validate the manufacture process, it is useful for the evaluation that tumorigenicity of
final products manufactured in the same manner and met the same quality standards istested at a certain number of local (e.g., under the retina or under the skin) in
immunodeficient animals by using the experimental system with a known detection
sensitivity. Final products derived from three donors or more, if possible, are tested.
Whenever necessary, analyses are conducted in a comprehensive manner by using the soft
agar colony formation assay or karyotype analysis, if scientifically relevant. However, thestudy of tumorigenicity may need to be revised, depending on new data from the clinicalapplications of the product.
As general principles on non-clinical safety, including tumorigenicity tests, the safety of
the final product and the safety of raw materials (iPS cells) are evaluated, in principle, in4

distinct from each other. For all non-clinical tests on retinal pigment epithelial cells derived
from autologous iPS cells, as functionally matured retinal pigment epithelial cells are used,
final products can be supposed to be equivalent to each other in terms of non-clinical safety
regardless of their donors or minor changes in the manufacture process, if the points toconsider in evaluation shown in this guidance are fully studied and the properties are wellcharacterized.
[2] Potency and performance assessment
As functionally matured cells are transplanted in the retinal pigment epithelialtransplantation, in principle, the cells are transplanted subretinally into animal models of
retinal pigment epithelial dysfunction, such as the Royal College of Surgeons (RCS) rats
and then their protective effect for retina as retinal pigment epithelial cells is confirmed.
[3] Other issues
If special handlings are needed for the insertion of the sheet and such, it is recommended
to confirm the items necessary and scientifically appropriate for the clinical application,such as the safety of the handlings and short-term local reactions following transplantation
with the handlings, in middle- and/or large-sized animals.
(3) Clinical studies (Clinical trials)[1] Indications
Diseases involving disorders in the retinal pigment epithelium
Diseases such as age-related macular degeneration, degenerative myopia, Stargardt’s
disease, traumatic injuries, and retinitis pigmentosa[2] Items for systemic monitoring
Adequate systemic screening of the presence of malignancies are recommended prior to
any surgical procedure to determine whether the tumor could be derived from transplanted
cells in case of development of a tumor in tissues other than eye after transplantation,. A
suitable period is established after transplant surgery, and attention is given in theeventuality that a tumor develops.
[3] Evaluation of transplantation treatment
For the diseases targeted in this guidance, the items for the evaluation of therapeutic effectare mainly categorized into 2 types: a) anatomical evaluation and b) visual performance
evaluation. Which should be used as evaluation items and the best timing to do so should beconsidered according to the targeted diseases and content of treatment. It seems plausiblethat control groups are selectedfrom the patients who seem adequate for the comparisonin consideration of the previous outcome of treatment and their control group, depending on
the study design: if patients who did not fully respond to conventional treatment (such as
anti-VEGF therapy in age-related macular degeneration) are the enrolled group, or if5patients meeting certain standards are used as controls regardless of the effectiveness of
their existing treatment are the ones to be enrolled, a comparison can be conducted with theresults of treated patients with conventional treatments in the past, or theose of the control
groups inof these past studies . In cases of hereditary degenerative diseases in which boththe eyes progress in the consistent time frame, the use of the contralateral eye as a control
may be considered appropriate.
The following is a summary of the current trend of the use of evaluation items in thespecialized field of ophthalmology. Tests used in the specialized field of ophthalmology
have remarkably progressed; suitable evaluation methods should be chosen for each test
design at any time.
a) Anatomical evaluation
Fundoscopic examination and diagnostic imaging, such as contrast imaging tests andretinal optical coherence tomography
Tests conducted in ophthalmology have shown remarkable progress in recent years.
For example, retinal optical coherence tomography (OCT) allows the observation of
tomographic

images of the ocular fundus in a non-invasive manner and with highresolution. It facilitates the detection of active exudative lesions such as those found inage-related macular degeneration, as well as the actual quantitative condition of theresidual visual cells after treatment, including dry types. Thus, OCT is a highly reliable
testing method that may be used for objective evaluation of the time-course evaluationof its protective effect on the retina as well. Therefore, use of diagnostic imaging
methods such as OCT is currently the most appropriate evaluation method to determine
the survival of transplanted cells and their effects. In addition, making assessments
based on contrast imaging tests and OCT is the most appropriate for the evaluation ofsafety (including rejection and tumor formation), because of their high sensitivity.
b) Visual function test
Visual function tests include assessment of visual acuity, retinal sensitivity, visual
field test, and electrophysiological testing
In terms of exudates during choroidal neovascularization, such as that found in
pigment epithelial failure in the macular region, as well as that observed in exudative
age-related macular degeneration, the pathogenesis of exudation includes progressive
degeneration of overlying photoreceptor cells in the macular region. Visualperformance depends on the condition of the retinal photoreceptor cells, and the main
purpose of the transplantation therapy is to prevent further progression of this macularvisual performance disorder (decrease in visual acuity) by supplying healthy pigment
epithelia under the residual retinal photoreceptor cells to protect the function of these6photoreceptors. Recovery of lost photoreceptor cells is currently impossible and cannot
be considered as a purpose for the pigment epithelial transplantation.
Central vision, or visual acuity, is generally used as an indicator of visual function,although influenced by the position of residual healthy retinal photoreceptor cells in thecentral region. Visual acuity, thus, is generally better when the residual retinal
photoreceptor cells are located closer to the central region. However, in age-related
macular degeneration, all retinal photoreceptor cells are not lost uniformly in aconcentric manner, and instead, they disappear in a random and disorderly manner.
Hence, the range of photoreceptor cells remaining in the macula area is not necessarily
correlated with visual acuity. Further, individual differences are also present in thesubjective perception of visual performance, such as the identification of important
points. Subjectively, the following types of dissociations may actually occur: “Duringvisual acuity tests, numbers are visible, but the subject does not have the impression of
being able to see” or “The visual acuity score is low, but surprisingly, there is no
disability.”
When the disease is treated early, a high number of photoreceptor cells are protected
at the central area, and generally, good visual acuity is maintained. On the other hand,when treatment is conducted at an advanced stage of the disease, the photoreceptor cells
in the central area are already lost, and therefore, the chances for any improvement invisual acuity may be significantly reduced. However, if the photoreceptor cells in thesurrounding areas can be protected, improvements may be achieved, such as a decreasein the central scotoma (the central blind area).
Therefore, depending on the timing and progression of the disease, when assessment
based on visual acuity alone is considered inappropriate for the evaluation of visualfunction, it is preferable to conduct a comprehensive assessment that includes visualacuity, retinal sensitivity or central area of vision, as well as indexes for macular andlocal reactivity and the range.
Dependingonthedisease,

if

local

analysescanbeperformed,

thenelectrophysiological tests may be conducted to assess the visual performance
objectively. In addition, in patients with bilateral impairment, if treatment is conductedon the eye with predominant visual acuity, then NEI VFQ-25 can also be used as anindicator in the evaluation of visual performance to examine the patient’s quality of life2).

6. References
1. Kuroda T, Yasuda S, Kusakawa S, Hirata N, Kanda Y, Suzuki K, Takahashi M, Nishikawa
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S, Kawamata S, Sato Y. Highly sensitive in vitro methods for detection of residual
undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells. PLoS
One. 2012;7(5):e37342.
2. Orr P, Rentz AM, Marfolis MK, Revicki DA, Dolan CM, Colman S, Fine JT, Bressler NM.
Validation of the National Eye Institute Visual Function Questionnaire-25 (NEI VFQ-25) inage related macular degeneration Invet. Ophthalmol.Vis. Sci. 2011;52:3354-3359.

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