fetal
Viability
Vi bility off human
h
f t l retina/RPE
ti /RPE sheets
h t after
ft 4 days
d y cold
ld storage
t
g
GC
GCL
NBL
Magdalene
g
J.
J
RPE
1 Department
D
t
t
20 m
Example of human retina with RPE
10-11 wks gestation
1,2
1
2
Seiler ,
Gabriel
1
3,1
3
Nistor , Norman
Overview
1
o o
Donor
#
Gestat
o a
Gestational
age
14.5
14 5
weeks
2
B kg
d
Background
p
off retinal sheets
Transplantation
GC
IP
IN
host
transplant
p
ON
RPE
Human fetal retina with RPE
transplanted to albino athymic
nude rat
rat, 8
8.5
5 months after surgery
E id
Evidence
off titissue viability
i bilit after
ft prolonged
l
d cold
ld storage
t
would
ld
widen
id the
th applicability
ppli bility off this
thi procedure
p
d
for
f clinical
li i l trials.
t i l The
Th
results would also be important
p
for development
p
of future trials
with hESC-derived
hESC derived tissue
tissue.
4
6
Abbreviations:
g
RD – retinal degeneration
GC(L) – ganglion cell layer
NBL – neuroblastic layer
IP – inner plexiform layer
IN – inner nuclear layer
ON – outer nuclear layer
y
RPE – retinal pigment epithelium
1 Tissue preparation
1.
p p
Permission to use fetal tissue for research was obtained from the Western Institution Review Board
Board, Norton Healthcare Research
ffi
d the
th hSCRO committee
itt off UC Irvine.
I i
office,
and
Retina together with its RPE was dissected from fetal eyes (received at 1 day (d) after abortion)
abortion). Eyes were placed into cold
t
d CO2-independent
i d
d t hibernation
hib
ti medium
di
(Hib
t E,
E Millipore
Milli
C
t
di ) with
ith B27 supplements
l
t (Gib
custom-made
(Hibernate
Custom
media)
(Gibco))
immediately after harvest
harvest. Eyecups were treated with dispase (Coll
(Coll. Res
Res. Inc
Inc.)) for 15-20
15 20 minutes at 37 ºC
C.
Di
Dissected
t d eye
i t pieces
(ranging
i size
i between
b t
2 to
t 7 mm2 )). Some
S
pieces
i
were fifixed
d iimmediately
di t ly ((= “f
“fresh”),
h”))
y cups
p were cutt into
pi
( gi g in
p
and the remaining pieces sucked up into flat plastic nozzles in hibernation medium in shipping tubes (see table below)
below).
1
2
3
Ti
Tissue
condition
diti
13-14 wks
# off p
pieces
i
1 eye
y (punctured)
(p
)
14.5 weeks
N/A
2 eyes; lost RPE off
d
one eye – nott used
6 pieces
2 eyes (good)
(
d)
5 pieces R eye,
3 pieces L eye
10 5 weeks
10.5
k
“fresh”
fresh
fixed
“1d
1d
shipping”
“2d
2d
shipping”
“3d
3d
shipping”
N/A
N/A
N/A
N/A
2
_
2
3
2 ((+ 1 llost)
t)
2
1
2
_
5
12.5 weeks
2 eyes (both punctured)
N/A
N/A
N/A
N/A
N/A
7 eyes (+ 1 not
used;; + 3 p
punctured
5 pieces L eye,
4 pieces
i
R eye
3
28 pieces
9
2 ((+ 1 lost))
3
_
6 (+
( 1 lost)
(2) rest of L eye; RPE
attached to retina but RPE
starting to dissolve in some
areas
(2) Piece in nozzle,
nozzle RPE
attached
(2) piece in nozzle,
nozzle RPE
attached
30
25
20
Box opened
for sample retrieval
(3) piece
i
i nozzle,
l
was in
RPE attached
(3) retina
ti + RPE;
RPE RPE
mostly intact
(1)
( ) Part of R macula,, retina
+ RPE
(1)
( ) Piece was in nozzle,,
RPE attached to retina
retina,
h l
some holes
(1)
( ) Piece was in nozzle;;
RPE with holes but still
tt h d piece
i
d
attached;
curved
(2) Rest of R eye after
cutting
g off p
pieces,, RPE
attached to retina,
retina small
areas of RPE dissolving
(1) Retina + RPE; part of
RPE folded
f ld d over
(2) Rest of L eye after
cutting
gp
pieces,, retina + RPE
(2) Retina + RPE
RPE, a bit
twisted
N/A
(2) Piece contains
macula,, RPE g
got ripped
pp
-piece disrupted,
disrupted only
smallll shreds
h d left
l ft
N/A
3-4
3 4 d after harvest
(3) retina + RPE
RPE, part of
RPE broken off
ff
200
180
160
60
Total number
140
of TUNEL + cells
per mm2
120
Th number
The
b off
apoptotic
p p
cells
with
i
ith
increases
storage
g time
***
***
**
100
80
60
40
0
A
Average
# off stained
t i d nuclei
l i iin positive
iti control:
t l
18,298 cells/mm2
10
5
5
5
0
10/29/08 10/29/08 10/30/08 10/30/08 10/30/08 10/30/08 10/31/08 10/31/08 10/31/08
0 21:25
6
12 9:25
18 15:25
24 21:25
30 3:25
36 9:25
42 15:25
48
15:25
3:25
hours
hours
Date Tim e
Date,
160
0
0
11/5/08
23:20
6
11/6/08
5:20
12
11/6/08
11:20
18
11/6/08
17:20
24
11/6/08
23:20
30
11/7/08
5:20
36
Date, Tim e
E
Experiment
i
t2
Outer neuroblastic layers
y
Total
T
t l number
b
+
of TUNEL cells
2
per mm
+
TUNEL cells
2
per mm
(inner layers)
11/5/08
17:20
11/7/08
11:20
42
hours
Date, Tim e
E
Experiment
i
t3
3 Histology
3.
gy and TUNEL staining
g (determining
(
g cell viability)
y)
Tissues were immersion-fixed
immersion fixed in 4% paraformaldehyde, infiltrated with 30% sucrose, and frozen in OCT.
10 μm cryostat cross
cross-sections
sections (3 sections/slide) were stained by an in situ cell death detection kit (Roche) based on fluorescent
TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Each staining set contained DNAse
DNAse-treated
treated
positive controls,
controls and negative controls (omission of reagents).
reagents) Sections were counterstained with DAPI (blue)
(blue).
Six to 28 fluorescent images/specimen were taken on a Nikon FXA microscope at 200x.
4 Cell
4.
C ll counting
ti
The number of TUNEL-stained (+) cells per mm2 and % TUNEL-stained cells (green) of total cells (blue) was counted in 10 μm
cryostat
y t t cross-sections,
ti
using
i g Diagnostic
Di g
ti Instruments
I t
t SPOT software,
ft
by
by counting
ti g in
i separate
p t channels.
h
l
This analysis was performed for
((1)) allll cells
ll in
i the
th field
fi ld ((allll llayers),
y )), and
d separately
p t ly
(2) for the inner retinal layers (containing differentiating neurons) and
((3)) outer
t neuroblastic
bl ti layers
l y
(th t would
((that
ld d
develop
l p iinto
t photoreceptors
ph t
pt
and
d other
th retinal
ti l cells).
ll )
Controls
for
o TUNEL
U
stain
t i
120
100
80
***
***
80
(outer
neuroblastic layer) 60
**
60
40
40
n.s.
n.s.
20
20
0
Average # of stained nuclei in inner layers in positive control:
7 343 cells/mm2
7,343
TUNEL+ cells
ll iin inner
i
layers
l
: between
b t
0
0.5
5 – 1.9
1 9 % off nuclei
l i
CONCLUSIONS
SO S
CO
C US
Intact sheets of fetal human retina with RPE can remain
viable
i bl for
f up
p to
t 3d
days
y after
ft harvesting
h
ti g with
ith careful
f l
dissection and storage
y after
Four
days
harvesting,
is
F
d
ft harvesting
h
ti g there
th
i significant
ig ifi
t tissue
ti
deterioration.
deterioration
d t i
ti
100
***
140
20
10
2-3
2
3d
shipping
hi i
Whole
Wh l section
ti
((all
ll llayers))
25
10
1 d after
ft harvest
h
t
2 d after
ft harvest
h
t
(2) Piece was in nozzle,
nozzle
RPE attached to retina,,
some holes
(1) Part of L macula
macula, retina + (1) Retina + RPE
RPE
n.s. = not significant
Box opened
for sample retrieval
fresh
1 d shipping
hi i
A
t i rate:
t increase
i
ith time
ti
Apoptosis
with
- difference between layers
y
30
15
Donor
#2
D
N/A
R i T
Retina
Transport 3
15
E
Experiment
i
t1
((1)) Piece came partially
p
y
out of nozzle
nozzle, damaged;
RPE came off
2d shipping
pp g
15
0
10/24/08 10/25/08 10/25/08 10/25/08 10/25/08 10/26/08 10/26/08 10/26/08 10/26/08 10/27/08 10/27/08
0
6
12 12:00
18 18:00
24 0:00
30 6:00
36 12:00
42 18:00
48 0:00
54 6:00
60
18:00
0:00
6:00
((1)) Retina + RPE, RPE
attached
Inner layers
y
((differentiating
g cells))
Temp
T
perature (°C)
Temperatu
T
ure (°°C)
Temp
T
peratture (°C)
20
f
((1)) Rest off R eye
y after
cutting pieces off; good
dissection; RPE attached
(3) rest of R eye after cutting (3) retina + RPE
RPE, part of
pieces off,
p
ff retina + RPE
RPE broken off
ff
3
Donor #6
(4) RPE in nozzle (rolled up)
7 (+
( 1 lost) 3 (+
( 1 lost)
R i T
Retina
Transport 2
25
nozzle lost;
(3) tissue not in nozzle,
TUNEL cells
TUNEL+
ll iin all
ll layers:
l y
between
b
0.3
0 3 – 0.9
0 9 % off all
ll nuclei
l i
2d shipping
30
N/A
(3) RPE came off after
cutting,
tti
only
l attached
tt h d att
ciliary body (rest of L eye)
** = p< 0.01
*** = p<
p 0.001
The temperature in the container was measured continuously by a temperature probe
probe.
R i T
Retina
Transport 1
(2) tissue in nozzle, flat – does
not look good ;
20
2 Temperature monitoring
2.
3d shipping
(2) Disrupted RPE during
dissection (piece of L eye)
“fresh”
“f
h” = 0d shipping
hippi g
all tissue was dissected
1 day
d y after
ft h
harvesting
ti g
5 pieces
p
Total tissue
( ) Tissue not in nozzle, rolled
(1)
up;
N/A
Donor #4
“3d
3d shipping”
shipping
hi i ”
4 d after
ft harvest
h
t
( ) Lost RPE during
(1)
g
dissection (whole R eye)
fresh
0d shipping = “fresh”
fresh
1d shipping
hi
ft h
harvestt
shipping
hi ii = 2d after
2d shipping
shipping = 3d after harvest
hi ii = 4d after
ft h
harvestt
3d shipping
shipping
_
1 eye
y (good)
(g
)
2 eyes
y (good)
(g
)
“fresh”
“ffresh
h”
1 d after
ft h
harvestt
p
g
Examples
of TUNEL Staining
2
Morphological
pp
M ph l gi l appearance
“1
1 d shipping”
shipping
“2
2 d shipping”
shipping
hi i ”
hi i ”
2 d after
ft h
harvestt 3 d after
ft harvest
h
t
(whole section)
12.5 weeks
12 weeks
12 weeks
k
3 (+1 lost)
_
4
6
12.5
12
5
weeks
Aramant & Seiler
Exp Eye Res 2002; 75:115
125
75:115-125
Methods
et ods
Donor Gestational
#
age (wks)
10.5
10
5
weeks
3
IP
IN
In a phase II clinical trial,
trial sheet transplants of retina
together with its RPE improved vision as tested by
EDTRS in 7 of 10 patients after one year
(Radtke et al.
al 2008)
Program # 695
B
Board
d # C0238
RESULTS
determine
retinal
To
T d
t
i viability
i bility off human
h
ti l sheets
h t
with its RPE after prolonged cold
storage
storage,
cold-storage,
including shipping in temperature
temperature-controlled
controlled
containers
t i
Retinal
R
ti l sheet
h t transplants
t
l t have
h
shown
h
to
t improve
i
p
visual
i
l responses
in
i four
f
different
diff
t rodent
d t retinal
ti l
g
models
degeneration
Robert B
B.
1
Aramant ,
off Anatomy
A t
and
d Neurobiology;
N
bi l
U i
University
it off California
C lif i att Irvine,
IIrvine
i
CA;
CA 2 currentt address:
dd
Department
D
t
t off Ph
Physical
i l Medicine
M di i & Rehabilitation,
Rehabilitation
R h bilit ti
University
U i
it off California
C lif i att Irvine
IIrvine,
i
CA;
CA
3 current address: California Stem Cell Inc
Inc., Irvine CA; 4 Retina Vitreous Resource Center,
Center Louisville,
Louisville KY
PURPOSE
Fetal retinal sheets can be gently implanted into the
subretinal space and restore a degenerating retina
D.
D
4
Radtke ,
0
Average # of stained nuclei in outer layers in positive control:
10 954 cells/mm2
10,954
TUNEL+ cells
ll in
i outer
t layers:
l
b
between
t
0.26
0 26 – 0.6
0 6 % off nuclei
l i
Cells in inner layers (differentiating) show a higher apoptosis rate
than cells in outer neuroblastic layer
y (developing
(
p g photoreceptors)
p
p
)
photoOuter neuroblastic layers (developing into photo
receptors)
pt
) show
h
lless apoptosis
p pt i than
th iinner retinal
ti l llayers.
layers
y
This may provide a time window for tissue testing which
o ld be useful
sef l in clinical trials
would
trials.
R f
References:
Aramant RB,
RB Seiler MJ.
MJ Transplanted sheets of human retina and retinal pigment
epithelium
ith li
d
develop
l normally
ll in
i nude
d rats.
t Exp
E Eye
E Res,
R
75(2)
75(2): 115-125
115 125 (2002)
(2002).
R dtk ND
Radtke
ND,, A
Aramantt RB,
RB, P
Petry
t y HM,
HM, G
Green PT
PT,, Pid
Pidwell
ll DJ
DJ,, S
Seiler
il MJ
MJ. Vi
Vision
i
Improvement in Retinal Degeneration Patients by Implantation of Retina Together
with
ith Retinal
R ti l Pi
Pigmentt Epithelium.
E ith li
A J Ophthalmol,
Am
O hth l l 146
146:172-182
146:172
172 182 (2008)
S il MJ
Seiler
MJ,, A
Aramantt RB
RB. C
Cell
ll replacement
pl
t and
d visual
i
l restoration
t
ti
b
by
y retinal
ti l sheet
h t
transplants Prog Retin Eye Res
transplants.
Res, 31:661-687
31:661 687 (2012)
(2012).
ACKNOWLEDGMENTS:
S pp t d by
Supported
by Lincy
Li y F
Foundation.
d ti
Author Disclosure Information (Commercial Relationship(s)):
Magdalene Seiler: Ocular Transplantation LLC: Code C (Consultant)
(Consultant), Code P (Patent);
Gabriel Nistor: Code N (No Commercial Relationship);
Norman D. Radtke: Code
C
N (No
(
Commercial
C
Relationship);
)
R b t Aramant
Robert
A
t FARVO:
FARVO Ocular
O l Transplantation
T
pl t ti LLC
LLC: C
Code
d E ((E
(Employment),
pl y
t)), C
Code
d P ((P
(Patent);
t t));