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Vi bilit fh ftl ti /RPE h t ft 4d ld t Vi bilit fh ftl ti /RPE h t ft 4d ld t GC Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage GCL Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage Viability of human fetal retina/RPE sheets after 4 days cold storage NBL NBL 12 31 4 1 Magdalene J Seiler 12 Gabriel Nistor 31 Norman D Radtke 4 Robert B Aramant 1 Magdalene J Seiler 1,2 Gabriel Nistor 3,1 Norman D Radtke 4 Robert B Aramant 1 Magdalene J. Seiler , Gabriel Nistor , Norman D. Radtke , Robert B. Aramant , 1 D t t fA t dN bi l Ui it f C lif i tI i CA 2 t dd D t t f Ph i lM di i & R h bilit ti Ui it f C lif i tI i CA RPE 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 # 695 20 m 3 current address: California Stem Cell Inc Irvine CA; 4 Retina Vitreous Resource Center Louisville KY Program # 695 B d # C0238 3 current address: California Stem Cell Inc., Irvine CA; 4 Retina Vitreous Resource Center, Louisville, KY Board # C0238 Example of human retina with RPE Example of human retina with RPE 10-11 wks gestation 10-11 wks gestation RESULTS RESULTS PURPOSE PURPOSE RESULTS RESULTS PURPOSE PURPOSE RESULTS RESULTS PURPOSE PURPOSE RESULTS RESULTS PURPOSE PURPOSE RESULTS RESULTS PURPOSE PURPOSE T dt i i bilit f h ti l h t 1 2 Overview Examples of TUNEL Staining To determine viability of human retinal sheets 1 2 Overview Examples of TUNEL Staining To determine viability of human retinal sheets 1 2 Overview Examples of TUNEL Staining 1 2 with its RPE after prolonged cold storage with its RPE after prolonged cold-storage, M hl i l D #2 with its RPE after prolonged cold storage, Morphological appearance Donor #4 Donor #6 Donor #2 including shipping in temperature controlled Donor Gestational “f h” “1 d hi i ” “2 d hi i ” “3d hi i ” Donor #4 Donor #6 Donor #2 including shipping in temperature-controlled # age “fresh” “1 d shipping” “2 d shipping” “3d shipping” including shipping in temperature controlled # age fresh 1d ft h t 1 d shipping 2d ft h t 2 d shipping 3d ft h t 3d shipping 4d ft h t ti 1 d after harvest 2 d after harvest 3 d after harvest 4 d after harvest containers (1) (1) containers (1) Lost RPE during (1) Tissue not in nozzle, rolled dissection (whole R eye) up; dissection (whole R eye) up; 14 5 (2) Disrupted RPE during (2) tissue in nozzle, flat – does 2 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 d B k d 2 weeks N/A N/A dissection (piece of L eye) not look good ; fresh Background Background weeks (3) RPE came off after (3) tissue not in nozzle lost; fresh 1d ft h t Background Background (3) RPE came off after tti l tt hd t (3) tissue not in nozzle, lost; 1 d after harvest Background Background cutting, only attached at ciliary body (rest of L eye) (4) RPE in nozzle (rolled up) f ciliary 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 cutting pieces off; good attached out of nozzle damaged; cutting pieces off; good dissection; RPE attached attached out of nozzle, damaged; RPE came off Fetal retinal sheets can be gently implanted into the dissection; RPE attached RPE came off Fetal 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 GC IP subretinal 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 host host IP IN subretinal space and restore a degenerating retina 3 weeks N/A attached to retina but RPE attached attached host host IN R ti l h t t l t h h t i weeks starting to dissolve in some IP Retinal sheet transplants have shown to improve starting to dissolve in some areas (3) i i l (3) ti RPE RPE IN Retinal 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 1d hi i transplant transplant visual responses in four different rodent retinal RPE attached mostly intact 1 d shipping ON degeneration models RPE attached mostly intact 1 d shipping 2d ft h t degeneration models 2 d after harvest RPE (1) Part of R macula, retina (1) Piece was in nozzle, (1) Piece was in nozzle; 2 d after harvest RPE In a phase II clinical trial sheet transplants of retina + RPE RPE attached to retina RPE with holes but still In a phase II clinical trial, sheet transplants of retina + RPE RPE attached to retina, hl RPE with holes but still tt hd i d Human fetal retina with RPE together with its RPE improved vision as tested by 12 5 some holes attached; piece curved N/A Human fetal retina with RPE transplanted to albino athymic together 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 surgery EDTRS 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 surgery EDTRS in 7 of 10 patients after one year weeks cutting off pieces, RPE RPE attached to retina, (Radtke et al 2008) attached to retina small some holes (Radtke et al. 2008) attached to retina, small areas of RPE dissolving some holes areas 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 of Evidence 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 ltil Th RPE RPE folded over 2 3d Aramant & Seiler widen the applicability of this procedure for clinical trials. The (2) Rest of L eye after (2) Piece contains (2) Retina + RPE a bit 2-3 d Aramant & Seiler Exp Eye Res 2002; 75:115-125 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 results would also be important for development of future trials 6 12 k N/A cutting pieces, retina + RPE macula, RPE got ripped twisted shipping with hESC derived tissue Abbreviations: 6 12 weeks -piece disrupted only shipping with hESC-derived tissue. Abbreviations : -piece disrupted, only ll h d l ft 3-4 d after harvest small 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 of GC(L) – ganglion cell layer (3) rest of R eye after cutting ff (3) retina + RPE, part of ff (3) retina + RPE, part of ff Methods Methods GC(L) ganglion cell layer NBL neuroblastic layer pieces off, retina + RPE RPE broken off RPE broken off Methods Methods NBL – neuroblastic layer IP inner plexiform layer Methods Methods IP – inner plexiform layer IN – inner nuclear layer ON – outer nuclear layer 3 RPE – retinal pigment epithelium 1 Tissue preparation A t i t i ith ti Controls 3 RPE retinal pigment epithelium 1. Tissue preparation Apoptosis rate: increase with time Controls 3 Apoptosis rate: increase with time for TUNEL 3 Permission to use fetal tissue for research was obtained from the Western Institution Review Board Norton Healthcare Research Apoptosis 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 layers ti office, and the hSCRO committee of UC Irvine. - difference between layers stain Retina together with its RPE was dissected from fetal eyes (received at 1 day (d) after abortion) Eyes were placed into cold - difference between layers stain Retina together with its RPE was dissected from fetal eyes (received at 1 day (d) after abortion). Eyes were placed into cold t d CO id d t hib ti di (Hib t E Milli C t di ) ith B27 l t (Gib ) custom-made CO 2 -independent hibernation medium (Hibernate E, Millipore Custom media) with B27 supplements (Gibco) 2 immediately after harvest Eyecups were treated with dispase (Coll Res Inc ) for 15-20 minutes at 37 ºC immediately after harvest. Eyecups were treated with dispase (Coll. Res. Inc.) for 15 20 minutes at 37 C. Di td ti t i ( i i i bt 2t 7 2 )S i fi di di t l ( “f h”) Dissected eye cups were cut into pieces (ranging in size between 2 to 7 mm 2 ). Some pieces were fixed immediately (= “fresh”), Wh l ti ( lll ) 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 “3d Donor # Gestational age (wks) Tissue condition # of pieces fresh fixed 1d shipping” 2d shipping” 3d shipping” fresh Th b f 0d shipping = “fresh” # age (wks) fixed shipping” shipping” shipping” 200 *** fresh 1d hi i The number of 0d shipping = fresh 1d hi i 2d ft h t CO C SO S CO C SO S 180 *** 1d shipping The number of 1d shipping = 2d after harvest CONCLUSIONS CONCLUSIONS 1 13-14 wks 1 eye (punctured) N/A N/A N/A N/A N/A 180 2d shipping apoptotic cells 2d shipping = 3d after harvest CONCLUSIONS CONCLUSIONS 160 *** 2d shipping 3d hi i apoptotic cells 2d shipping 3d after harvest 3d hi i 4d ft h t CONCLUSIONS CONCLUSIONS 2 eyes; lost RPE off 3 (+1 lost) Total number 3d shipping i ith 3d shipping = 4d after harvest CONCLUSIONS CONCLUSIONS 2 14.5 weeks 2 eyes; lost RPE off t d 6 pieces 2 3 (+1 lost) of TUNEL + cells 140 ** increases with 2 14.5 weeks one eye – not used 6 pieces 2 _ _ of TUNEL cells per mm 2 120 increases with per mm 120 (whole section) storage time 3 10 5 k 2 ( d) 5 pieces R eye, 2 3 2( 1l t) Intact sheets of fetal human retina with RPE can remain 100 (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 remain 80 “f h” 0d hi i 3 pieces L eye Intact sheets of fetal human retina with RPE can remain 80 “fresh” = 0d shipping i bl f t 3d ft h ti ith fl 60 all tissue was dissected 4 12.5 weeks 1 eye (good) 5 pieces 2 1 2 viable for up to 3 days after harvesting with careful 40 all tissue was dissected 1d ft h ti viable for up to 3 days after harvesting with careful 40 1 day after harvesting 20 5 12.5 weeks 2 eyes (both punctured) N/A N/A N/A N/A N/A dissection and storage 20 5 12.5 weeks 2 eyes (both punctured) N/A N/A N/A N/A N/A dissection and storage 0 ** dissection and storage A # f ti d l ii iti t l ** = p< 0.01 5 pieces L eye, Average # of stained nuclei in positive control: 2 *** = p< 0.001 6 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 ti 18,298 cells/mm 2 p 0.001 4 pieces R eye Four days after harvesting there is significant tissue TUNEL ll i ll l b 03 09% f ll li n.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 not dt i ti Total tissue 7 eyes (+ 1 not 28 pieces 9 6 (+ 1 lost) 7 (+ 1 lost) 3 (+ 1 lost) deterioration Total tissue used; + 3 punctured 28 pieces 9 6 ( 1 lost) 7 ( 1 lost) 3 ( 1 lost) deterioration. 2 Temperature monitoring Outer neuroblastic layers (developing into photo 2. Temperature monitoring Outer neuroblastic layers (developing into photo- 2. Temperature monitoring Outer neuroblastic layers (developing into photo Inner layers (differentiating cells) Outer neuroblastic layers The temperature in the container was measured continuously by a temperature probe t ) h l t i th i ti ll Inner layers (differentiating cells) Outer neuroblastic layers The temperature in the container was measured continuously by a temperature probe. receptors) show less apoptosis than inner retinal layers receptors) show less apoptosis than inner retinal layers. R i T 1 R i T 2 R i T 3 Retina Transport 1 3d shipping Retina Transport 2 2d shipping Retina Transport 3 2d shipping 30 (°C) 3d shipping 30 °C) 2d shipping 30 °C) 160 100 This may provide a time window for tissue testing which 25 ture 25 ure (° 25 ure ( 160 *** Ttl b 100 This may provide a time window for tissue testing which 25 perat 25 eratu 25 perat 140 Total number + This may provide a time window for tissue testing which 20 Temp 20 Temp 20 Temp Box opened + 140 of TUNEL + cells 2 o ld be sef l in clinical trials 15 T 15 T Box opened for sample retrieval 15 T Box opened for sample retrieval TUNEL + cells 2 120 per mm 2 80 *** would be useful in clinical trials 10 10 for sample retrieval 10 per mm 2 120 *** would be useful in clinical trials. 10 10 10 (inner layers) 100 *** (outer 5 5 5 100 neuroblastic layer) 60 0 0 0 80 ** 10/24/08 18:00 10/25/08 0:00 10/25/08 6:00 10/25/08 12:00 10/25/08 18:00 10/26/08 0:00 10/26/08 6:00 10/26/08 12:00 10/26/08 18:00 10/27/08 0:00 10/27/08 6:00 0 6 12 18 24 30 36 42 48 54 60 10/29/08 15:25 10/29/08 21:25 10/30/08 3:25 10/30/08 9:25 10/30/08 15:25 10/30/08 21:25 10/31/08 3:25 10/31/08 9:25 10/31/08 15:25 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 80 ** Rf 18:00 0:00 6:00 12:00 18:00 0:00 6:00 12:00 18:00 0:00 6:00 hours 15:25 21:25 3:25 9:25 15:25 21:25 3:25 9:25 15:25 Date, Time hours 17:20 23:20 5:20 11:20 17:20 23:20 5:20 11:20 Date, Time hours 60 40 n.s. References: Date, Time Date, Time Date, Time E i t1 E i t2 E i t3 60 References: Experiment 1 Experiment 2 Experiment 3 40 n.s. Aramant RB Seiler MJ Transplanted sheets of human retina and retinal pigment Experiment 1 Experiment 2 Experiment 3 40 20 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 tRB P t HM G PT Pid ll DJ S il MJ Vi i 3. Histology and TUNEL staining (determining cell viability) 0 0 Radtke ND, Aramant RB, Petry HM, Green PT, Pidwell DJ, Seiler MJ. Vision 0 0 Improvement in Retinal Degeneration Patients by Implantation of Retina Together Tissues 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 JO 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/mm 2 Average # of stained nuclei in outer layers in positive control: 10 954 cells/mm 2 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/mm 2 10,954 cells/mm 2 with Retinal Pigment Epithelium. Am J Ophthalmol, 146:172 182 (2008) S il MJ A tRB C ll l t d i l t ti b ti l h t TUNEL 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 bt 05 19% f li TUNEL + ll i t l bt 0 26 06% f li transplants 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 db Li F d ti 4 C ll ti ACKNOWLEDGMENTS: Supported by Lincy Foundation. 4. Cell counting 4. 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 mm 2 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 μm tt 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); This analysis was performed for Gabriel Nistor: Code N (No Commercial Relationship); This analysis was performed for (1) ll ll i th fi ld ( ll l ) d tl 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); (2) for the inner retinal layers (containing differentiating neurons) and Norman D. Radtke: Code N (No Commercial Relationship); Rb tA 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 it ht 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).
