Vi bilit f h f t l ti /RPE h t ft 4 d ld tVi bilit f h f t l ti /RPE h t ft 4 d ld tGC Viability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storageGCL Viability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storageViability of human fetal retina/RPE sheets after 4 days cold storagey y gy y gy y gy y gNBLNBL

1 2 3 1 4 1Magdalene J Seiler1 2 Gabriel Nistor3 1 Norman D Radtke4 Robert B Aramant1Magdalene J Seiler1,2 Gabriel Nistor3,1 Norman D Radtke4 Robert B Aramant1Magdalene J. Seiler , Gabriel Nistor , Norman D. Radtke , Robert B. Aramant , g , , , ,1 D t t f A t d N bi l U i it f C lif i t I i CA 2 t dd D t t f Ph i l M di i & R h bilit ti U i it f C lif i t I i CARPE 1 Department of Anatomy and Neurobiology; University of California at Irvine CA; 2 current address: Department of Physical Medicine & Rehabilitation University of California at Irvine CA;RPE Department of Anatomy and Neurobiology; University of California at Irvine, CA; current address: Department of Physical Medicine & Rehabilitation, University of California at Irvine, CA; Program # 69520 m

3 current address: California Stem Cell Inc Irvine CA; 4 Retina Vitreous Resource Center Louisville KYProgram # 695B d # C02383 current address: California Stem Cell Inc., Irvine CA; 4 Retina Vitreous Resource Center, Louisville, KY Board # C0238Example of human retina with RPEExample of human retina with RPE

10-11 wks gestation10-11 wks gestation

RESULTSRESULTSPURPOSEPURPOSE RESULTSRESULTSPURPOSEPURPOSE RESULTSRESULTSPURPOSEPURPOSE RESULTSRESULTSPURPOSEPURPOSE RESULTSRESULTSPURPOSEPURPOSET d t i i bilit f h ti l h t 1 2Overview Examples of TUNEL StainingTo determine viability of human retinal sheets 1 2Overview Examples of TUNEL StainingTo determine viability of human retinal sheets 1 2Overview Examples of TUNEL Stainingy 1 2 p gwith its RPE after prolonged cold storagewith its RPE after prolonged cold-storage, M h l i l D #2with its RPE after prolonged cold storage, Morphological appearance Donor #4 Donor #6 Donor #2including shipping in temperature controlled Donor Gestational

p g pp“f h” “1 d hi i ” “2 d hi i ” “3d hi i ”

Donor #4 Donor #6 Donor #2including shipping in temperature-controlled o o

#Gestat o a

age “fresh” “1 d shipping” “2 d shipping” “3d shipping”including shipping in temperature controlled # age fresh 1 d ft h t

1 d shipping2 d ft h t

2 d shipping3 d ft h t

3d shipping4 d ft h tt i 1 d after harvest 2 d after harvest 3 d after harvest 4 d after harvestcontainers (1) (1)containers (1) Lost RPE during (1) Tissue not in nozzle, rolled ( ) g

dissection (whole R eye)( )up;dissection (whole R eye) up;

14 5 (2) Disrupted RPE during (2) tissue in nozzle, flat – does2 14.5

N/A N/A(2) Disrupted RPE during dissection (piece of L eye)

(2) tissue in nozzle, flat does not look good ; fresh

B k dB k d2 weeks

N/A N/Adissection (piece of L eye) not look good ; freshBackgroundBackground weeks

(3) RPE came off after (3) tissue not in nozzle lost;fresh

1 d ft h tBackgroundBackground (3) RPE came off after tti l tt h d t

(3) tissue not in nozzle, lost; 1 d after harvestBackgroundBackground cutting, only attached at ggciliary body (rest of L eye) (4) RPE in nozzle (rolled up)

fciliary body (rest of L eye) (4) RPE in nozzle (rolled up)(1) f f (1) (1)Transplantation of retinal sheets (1) Rest of R eye after (1) Retina + RPE, RPE (1) Piece came partially Transplantation of retinal sheets ( ) ycutting pieces off; good

( )attached

( ) p yout of nozzle damaged;p cutting pieces off; good

dissection; RPE attachedattached out of nozzle, damaged;

RPE came offFetal retinal sheets can be gently implanted into the dissection; RPE attached RPE came offFetal retinal sheets can be gently implanted into the 10 5 (2) rest of L eye; RPE (2) Piece in nozzle RPE (2) piece in nozzle RPE

GCIPsubretinal space and restore a degenerating retina 3 10.5

N/A(2) rest of L eye; RPE (2) Piece in nozzle, RPE (2) piece in nozzle, RPE hosthostIP

INsubretinal space and restore a degenerating retina 3 weeksN/Aattached to retina but RPE attached attached

hosthostIN

R ti l h t t l t h h t iweeks starting to dissolve in someIP

Retinal sheet transplants have shown to improve starting to dissolve in some areas (3) i i l (3) ti RPE RPE

INRetinal sheet transplants have shown to improve i l i f diff t d t ti l

areas (3) piece was in nozzle, (3) retina + RPE; RPE 1 d hi itransplanttransplantvisual responses in four different rodent retinal RPE attached mostly intact 1 d shippingpp

ONpdegeneration models

RPE attached mostly intact 1 d shipping2 d ft h tdegeneration models 2 d after harvestRPEg

(1) Part of R macula, retina (1) Piece was in nozzle, (1) Piece was in nozzle; 2 d after harvestRPE

In a phase II clinical trial sheet transplants of retina( ) ,+ RPE

( ) ,RPE attached to retina

( ) ;RPE with holes but stillIn a phase II clinical trial, sheet transplants of retina + RPE RPE attached to retina,

h lRPE with holes but still

tt h d i dHuman fetal retina with RPEtogether with its RPE improved vision as tested by 12 5 some holes attached; piece curved

N/AHuman fetal retina with RPE

transplanted to albino athymictogether with its RPE improved vision as tested by 4 12.5 N/A(2) Rest of R eye after (2) Piece was in nozzle

transplanted to albino athymic nude rat 8 5 months after surgeryEDTRS in 7 of 10 patients after one year 4 weeks (2) Rest of R eye after (2) Piece was in nozzle, nude rat, 8.5 months after surgeryEDTRS in 7 of 10 patients after one year weeks cutting off pieces, RPE RPE attached to retina,

(Radtke et al 2008)g p ,

attached to retina small,

some holes(Radtke et al. 2008) attached to retina, small areas of RPE dissolving

some holesareas of RPE dissolving

E id f ti i bilit ft l d ld t ld (1) Part of L macula retina + (1) Retina + RPE (1) Retina + RPE; part ofEvidence of tissue viability after prolonged cold storage would (1) Part of L macula, retina + RPE

(1) Retina + RPE (1) Retina + RPE; part of RPE f ld d

Evidence of tissue viability after prolonged cold storage would id th li bilit f thi d f li i l t i l Th RPE RPE folded over 2 3 dAramant & Seilerwiden the applicability of this procedure for clinical trials. The

(2) Rest of L eye after (2) Piece contains (2) Retina + RPE a bit2-3 d Aramant & Seiler

Exp Eye Res 2002; 75:115-125pp y p

results would also be important for development of future trials (2) Rest of L eye after (2) Piece contains (2) Retina + RPE, a bit hi i

Exp Eye Res 2002; 75:115 125results would also be important for development of future trials 6 12 k N/Acutting pieces, retina + RPE macula, RPE got ripped twisted shippingp p

with hESC derived tissueAbbreviations: 6 12 weeks g p , , g pp

-piece disrupted onlyshippingwith hESC-derived tissue.

Abbreviations: -piece disrupted, only ll h d l ft 3-4 d after harvestsmall shreds left 3 4 d after harvest

RD – retinal degeneration (3) rest of R eye after cutting (3) retina + RPE part of (3) retina + RPE part ofgGC(L) – ganglion cell layer (3) rest of R eye after cutting

ff(3) retina + RPE, part of

ff(3) retina + RPE, part of

ffMethodsMethodsGC(L) ganglion cell layerNBL neuroblastic layer pieces off, retina + RPE RPE broken off RPE broken offMethodsMethods NBL – neuroblastic layerIP inner plexiform layer

pMethodsMethods IP – inner plexiform layeret odset odsIN – inner nuclear layerON – outer nuclear layer

3y

RPE – retinal pigment epithelium1 Tissue preparation A t i t i ith ti Controls3RPE retinal pigment epithelium1. Tissue preparation Apoptosis rate: increase with time Controls 3p p Apoptosis rate: increase with time for TUNEL3Permission to use fetal tissue for research was obtained from the Western Institution Review Board Norton Healthcare ResearchApoptosis rate: increase with time for TUNEL Permission to use fetal tissue for research was obtained from the Western Institution Review Board, Norton Healthcare Research

ffi d th hSCRO itt f UC I i difference between layerso U

t ioffice, and the hSCRO committee of UC Irvine. - difference between layers stainRetina together with its RPE was dissected from fetal eyes (received at 1 day (d) after abortion) Eyes were placed into cold - difference between layers stainRetina together with its RPE was dissected from fetal eyes (received at 1 day (d) after abortion). Eyes were placed into coldt d CO i d d t hib ti di (Hib t E Milli C t di ) ith B27 l t (Gib )

ycustom-made CO2-independent hibernation medium (Hibernate E, Millipore Custom media) with B27 supplements (Gibco) 2immediately after harvest Eyecups were treated with dispase (Coll Res Inc ) for 15-20 minutes at 37 ºCimmediately after harvest. Eyecups were treated with dispase (Coll. Res. Inc.) for 15 20 minutes at 37 C.Di t d t i t i ( i i i b t 2 t 7 2 ) S i fi d i di t l ( “f h”)Dissected eye cups were cut into pieces (ranging in size between 2 to 7 mm2 ). Some pieces were fixed immediately (= “fresh”),

Wh l ti ( ll l )y p p ( g g ) p y ( )

and the remaining pieces sucked up into flat plastic nozzles in hibernation medium in shipping tubes (see table below) Whole section (all layers)and the remaining pieces sucked up into flat plastic nozzles in hibernation medium in shipping tubes (see table below). Whole section (all layers)Donor Gestational Ti diti # f i “fresh” “1d “2d “3dDonor

#Gestational age (wks) Tissue condition # of pieces fresh

fixed1d

shipping”2d

shipping”3d

shipping” fresh Th b f0d shipping = “fresh”# age (wks) p fixed shipping” shipping” shipping” 200***

fresh1d hi i The number of0d shipping = fresh1d hi i 2d ft h t

CO C S O SCO C S O S180***1d shipping The number of 1d shipping = 2d after harvest

CONCLUSIONSCONCLUSIONS1 13-14 wks 1 eye (punctured) N/A N/A N/A N/A N/A 1802d shipping apoptotic cells2d shipping = 3d after harvest CONCLUSIONSCONCLUSIONSy (p )

160 ***2d shipping3d hi i

apoptotic cells 2d shipping 3d after harvest3d hi i 4d ft h t CONCLUSIONSCONCLUSIONS2 eyes; lost RPE off 3 (+1 lost) Total number

603d shippingp p

i ith3d shipping = 4d after harvest CONCLUSIONSCONCLUSIONS2 14.5 weeks 2 eyes; lost RPE off

t d 6 pieces 2 3 (+1 lost)of TUNEL + cells 140 ** increases with CO C US O SCO C US O S2 14.5 weeks one eye – not used 6 pieces 2 _ _ of TUNEL cells

per mm2 120increases with

per mm 120(whole section) storage time

3 10 5 k 2 ( d) 5 pieces R eye, 2 3 2 ( 1 l t) Intact sheets of fetal human retina with RPE can remain100(whole section) storage time 3 10.5 weeks 2 eyes (good) 5 pieces R eye,

3 pieces L eye 2 3 2 (+ 1 lost) _ Intact sheets of fetal human retina with RPE can remain80“f h” 0d hi ig

3 pieces L eye Intact sheets of fetal human retina with RPE can remain 80“fresh” = 0d shipping

i bl f t 3 d ft h ti ith f l60pp g

all tissue was dissected4 12.5 weeks 1 eye (good) 5 pieces 2 1 2 viable for up to 3 days after harvesting with careful40all tissue was dissected 1 d ft h ti

y (g ) p _ viable for up to 3 days after harvesting with careful 401 day after harvesting p y g20

y g

5 12.5 weeks 2 eyes (both punctured) N/A N/A N/A N/A N/A dissection and storage20

5 12.5 weeks 2 eyes (both punctured) N/A N/A N/A N/A N/A dissection and storage0** dissection and storage

A # f t i d l i i iti t l** = p< 0.01

5 pieces L eye, Average # of stained nuclei in positive control: 2

*** = p< 0.0016 12 weeks 2 eyes (good)

5 pieces L eye,4 i R

3 2 (+ 1 lost) 3 F d ft h ti th i i ifi t ti18,298 cells/mm2 p 0.001

y (g )4 pieces R eye

( ) _ Four days after harvesting there is significant tissueTUNEL ll i ll l b 0 3 0 9 % f ll l in.s. = not significant Four days after harvesting, there is significant tissue TUNEL+ cells in all layers: between 0.3 – 0.9 % of all nuclei

7 eyes (+ 1 noty g g

d t i tiy

Total tissue 7 eyes (+ 1 not 28 pieces 9 6 (+ 1 lost) 7 (+ 1 lost) 3 (+ 1 lost) deteriorationTotal tissue used; + 3 punctured 28 pieces 9 6 ( 1 lost) 7 ( 1 lost) 3 ( 1 lost) deterioration.; p

2 Temperature monitoring Outer neuroblastic layers (developing into photo2. Temperature monitoring Outer neuroblastic layers (developing into photo-2. Temperature monitoring Outer neuroblastic layers (developing into photoInner layers (differentiating cells) Outer neuroblastic layersThe temperature in the container was measured continuously by a temperature probe t ) h l t i th i ti l lInner layers (differentiating cells) Outer neuroblastic layersThe temperature in the container was measured continuously by a temperature probe. receptors) show less apoptosis than inner retinal layersy ( g ) y receptors) show less apoptosis than inner retinal layers.

R i T 1 R i T 2 R i T 3p ) p p y

Retina Transport 13d shipping Retina Transport 22d shipping Retina Transport 32d shipping30(°C

) 3d shipping30

°C)

2d shipping30°C

)

pp g160 100 This may provide a time window for tissue testing which25tu

re

25ure

(°

25ure

( 160 *** T t l b100 This may provide a time window for tissue testing which25

pera

t 25

erat

u 25

pera

t

140Total number

+ This may provide a time window for tissue testing which 20

Tem

p 20

Tem

p 20

Tem

p

Box opened +140 of TUNEL + cells

2

o ld be sef l in clinical trials15

T

15

T Box opened for sample retrieval 15

T Box opened for sample retrieval TUNEL + cells

2 120per mm2 80 *** would be useful in clinical trials10 10

for sample retrieval

10per mm2 120

*** would be useful in clinical trials.10 10 10(inner layers)

100*** (outer

5 5 5 100 neuroblastic layer) 600 0 0 80 **10/24/0818:00

10/25/080:00

10/25/086:00

10/25/0812:00

10/25/0818:00

10/26/080:00

10/26/086:00

10/26/0812:00

10/26/0818:00

10/27/080:00

10/27/086:000 6 12 18 24 30 36 42 48 54 60 10/29/08

15:2510/29/08

21:2510/30/08

3:2510/30/08

9:2510/30/08

15:2510/30/08

21:2510/31/08

3:2510/31/08

9:2510/31/08

15:250 6 12 18 24 30 36 42 48 11/5/0817:20

11/5/0823:20

11/6/085:20

11/6/0811:20

11/6/0817:20

11/6/0823:20

11/7/085:20

11/7/0811:200 6 12 18 24 30 36 42 80 **

R f18:00 0:00 6:00 12:00 18:00 0:00 6:00 12:00 18:00 0:00 6:00

Date Timehours15:25 21:25 3:25 9:25 15:25 21:25 3:25 9:25 15:25

Date, Timehours17:20 23:20 5:20 11:20 17:20 23:20 5:20 11:20

Date, Timehours

60 40 n.s. References:Date, Time Date, Time Date, Time

E i t 1 E i t 2 E i t 360 References:

Experiment 1 Experiment 2 Experiment 3 40 n.s. Aramant RB Seiler MJ Transplanted sheets of human retina and retinal pigmentExperiment 1 Experiment 2 Experiment 3 4020

Aramant RB, Seiler MJ. Transplanted sheets of human retina and retinal pigment ith li d l ll i d t E E R 75(2) 115 125 (2002)20

20 epithelium develop normally in nude rats. Exp Eye Res, 75(2): 115-125 (2002).3 Histology and TUNEL staining (determining cell viability) 20 epithelium develop normally in nude rats. Exp Eye Res, 75(2): 115 125 (2002).

R dtk ND A t RB P t HM G PT Pid ll DJ S il MJ Vi i3. Histology and TUNEL staining (determining cell viability)0 0 Radtke ND, Aramant RB, Petry HM, Green PT, Pidwell DJ, Seiler MJ. Vision gy g ( g y)0 0 , , y , , ,

Improvement in Retinal Degeneration Patients by Implantation of Retina TogetherTissues were immersion-fixed in 4% paraformaldehyde, infiltrated with 30% sucrose, and frozen in OCT. Average # of stained nuclei in inner layers in positive control: Average # of stained nuclei in outer layers in positive control:Improvement in Retinal Degeneration Patients by Implantation of Retina Together

ith R ti l Pi t E ith li A J O hth l l 146 172 182 (2008)Tissues were immersion fixed in 4% paraformaldehyde, infiltrated with 30% sucrose, and frozen in OCT. 10 μm cryostat cross sections (3 sections/slide) were stained by an in situ cell death detection kit (Roche) based on fluorescent

Average # of stained nuclei in inner layers in positive control: 7 343 cells/mm2

Average # of stained nuclei in outer layers in positive control: 10 954 cells/mm2 with Retinal Pigment Epithelium. Am J Ophthalmol, 146:172-182 (2008)10 μm cryostat cross-sections (3 sections/slide) were stained by an in situ cell death detection kit (Roche) based on fluorescent 7,343 cells/mm2 10,954 cells/mm2 with Retinal Pigment Epithelium. Am J Ophthalmol, 146:172 182 (2008)

S il MJ A t RB C ll l t d i l t ti b ti l h tTUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Each staining set contained DNAse-treated Seiler MJ, Aramant RB. Cell replacement and visual restoration by retinal sheet TUNEL staining (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Each staining set contained DNAse treated positive controls and negative controls (omission of reagents) Sections were counterstained with DAPI (blue) TUNEL+ ll i i l b t 0 5 1 9 % f l i TUNEL+ ll i t l b t 0 26 0 6 % f l i

, p ytransplants Prog Retin Eye Res 31:661 687 (2012)positive controls, and negative controls (omission of reagents). Sections were counterstained with DAPI (blue). TUNEL+ cells in inner layers : between 0.5 – 1.9 % of nuclei TUNEL+ cells in outer layers: between 0.26 – 0.6 % of nuclei transplants. Prog Retin Eye Res, 31:661-687 (2012).

Six to 28 fluorescent images/specimen were taken on a Nikon FXA microscope at 200x.Six to 28 fluorescent images/specimen were taken on a Nikon FXA microscope at 200x.

ACKNOWLEDGMENTS: S t d b Li F d ti4 C ll ti ACKNOWLEDGMENTS: Supported by Lincy Foundation.4. Cell counting pp y y4. Cell counting Cells in inner layers (differentiating) show a higher apoptosis rate Author Disclosure Information (Commercial Relationship(s)):The number of TUNEL-stained (+) cells per mm2 and % TUNEL-stained cells (green) of total cells (blue) was counted in 10 μm Cells in inner layers (differentiating) show a higher apoptosis rate Author Disclosure Information (Commercial Relationship(s)): The number of TUNEL-stained (+) cells per mm and % TUNEL-stained cells (green) of total cells (blue) was counted in 10 μmt t ti i Di ti I t t SPOT ft b ti i t h l than cells in outer neuroblastic layer (developing photoreceptors) Magdalene Seiler: Ocular Transplantation LLC: Code C (Consultant) Code P (Patent);cryostat cross-sections, using Diagnostic Instruments SPOT software, by counting in separate channels. than cells in outer neuroblastic layer (developing photoreceptors) Magdalene Seiler: Ocular Transplantation LLC: Code C (Consultant), Code P (Patent); y g g y g p

This analysis was performed fory ( p g p p )

Gabriel Nistor: Code N (No Commercial Relationship);This analysis was performed for (1) ll ll i th fi ld ( ll l ) d t l Gabriel Nistor: Code N (No Commercial Relationship);

C ( C )(1) all cells in the field (all layers), and separately

Norman D. Radtke: Code N (No Commercial Relationship);( ) ( y ), p y(2) for the inner retinal layers (containing differentiating neurons) and Norman D. Radtke: Code N (No Commercial Relationship);

R b t A t FARVO O l T l t ti LLC C d E (E l t) C d P (P t t)(2) for the inner retinal layers (containing differentiating neurons) and (3) t bl ti l (th t ld d l i t h t t d th ti l ll ) Robert Aramant FARVO: Ocular Transplantation LLC: Code E (Employment), Code P (Patent); (3) outer neuroblastic layers (that would develop into photoreceptors and other retinal cells). p ( p y ), ( );( ) y ( p p p )