Stem Cells for Retinal Disease

Transcription

1 Stem Cells for Retinal Disease Henry J. Klassen, MD, PhD Associate Professor Director, Stem Cell and Retinal Regeneration Program Gavin Herbert Eye Institute University of California, Irvine

2 Retinal Degenerations: What the doctor sees What the patient sees Retinitis Pigmentosa (RP) Hereditary Age-Related Macular Degeneration (AMD) Aging Gavin Herbert Eye Institute

3 Important commonality: Both = loss of rods & cones (photoreceptors) Gavin Herbert Eye Institute

4 How to repair the retina? Retinal Progenitor Cells (RPCs) Cells only found in the developing eye RPCs are immature cells Like stem cells, but specific to retina Have favorable safety profile Can be grown in lab, banked, transplanted Migrate to right location Become new retinal cells Integrate into existing circuitry RPCs normally generate the retina Are able to regenerate it, too

5 RPCs from fluorescent green mice RPCs Molecular analysis Gavin Herbert Eye Institute

6 Transplant to eye of blind rodents Look for green fluorescent cells: Bipolar cells Rod photoreceptors Green cells are RPCs from transplant, yet look like normal retinal cells that have integrated into precisely the right spot. Gavin Herbert Eye Institute

7 RPCs also rescue photoreceptors in the degenerating retina RPCs Sham injection Untreated Gavin Herbert Eye Institute 7

8 Grafted RPCs enhance light sensitivity in mice with retinal degeneration (rho -/-) EGFP Sham Retinal Progenitor Transplants Wheel- Running Activity Dark 20 ft candle 10 ft candle 5 ft candle 2.5 candle Dark High Lower Lower Lowest Light Levels Klassen et al (2004) IOVS

9 In vivo CNS stem cells grafted to the kidney capsule (non-privileged site) survive in naïve allorecipients CNS stem cells grafted to the kidney capsule survive in na ve allorecipients Hori, et al. In mice, CNS stem cells are not rejected anywhere

10 RPC transplantation in blind pigs GFP Rhodopsin Recoverin New rod photoreceptors Green fluorescent pigs No immune suppression Gavin Herbert Eye Institute

11 Transplantation of RPCs to blind cats Red fluorescent cat New retinal Mueller cell No immune suppression Abyssinian Dystrophic, CEP290 11

12 Transplantation of RCPCs to blind cats Substantial integration of donor cells from subretinal injection into overlying retina. No immune suppression Anti-RFP Anti-rod opsin Anti-vimentin

13 Human RPCs Human RPCs Molecular analyses Gavin Herbert Eye Institute

14 Make photoreceptors after subretinal injection in rd1mice with rapid retinal degeneration Jiang et al. Schepens Eye Research Inst.

15 Of greater clinical relevance, human RPCs also rescue photoreceptors, restore vision in RCS rats * Cell Sham untreated Optomotor Response Luminance threshold Untreated littermate Human RPC transplant 15

16 A A RPE ONL INL RGC Subretinal Injection of hrpcs Substantial local rescue 50µm B B No rescue away from the site 400µm Subretinal injection of retinal progenitor cells (RPC) in RCS rat, viewed at P100: Left-montage image of cresyl violet stained retinal section. A: high power image in outline A showing injection site (star), donor cells (arrows) and ONL preservation. B: high power image in outline B showing a single layer of ONL (arrows) in area away from the graft.

17 A A RPE INL ONL RG C 50µm Subretinal Injection of vehicle (sham) Also shows local rescue (moderate) B B RGC No rescue away from the site INL 400µm RPE Subretinal injection of vehicle in RCS rat, viewed at P100: Left-montage image of cresyl violet stained section. A: high power Image of outline A showing injection site (star) and localized ONL rescue. B: high power image of outline B; showing a single layer of ONL (arrows) at area away from injection site.

18 A A RPE ONL INL RGC Intravitreal Injection of hrpcs Substantial pan-retinal rescue 50µm B B INL RGC Rescue is everywhere 400µm ONL RPE Intravitreal injection of RPC in RCS rat, viewed at P100. Left-montage image of cresyl violet stained section showing ONL rescue over the whole retinal. A: high power of outline A showing 5-6 layers of ONL.B: high power image of outline B showing ONL preservation.

19 A A RPE INL RGC 50µm Intravitreal Injection of vehicle (sham) No discernable rescue* B B INL RGC *(There will always be a nonspecific effect at initial needle entry point) 400µm RPE Intravitreal injection of carry medium in RCS rat, viewed at P100. Left-montage image of cresyl violet stained section. A&B: high power of outline A &B showing a single layer of photoreceptors (arrows)

20 Summary: Proof-of-Concept in RCS Rats Subretinal hrpcs does generate local rescue (~10% of retinal area) Subretinal sham = some local rescue, but less Intravitreal hrpcs = pan-retinal rescue (100% of retinal area) Intravitreal sham = negligible The anatomical rescue results in functional rescue: 1) Optomotor Response 2) Luminance Threshold (Brain)

21 Human Retinal Progenitor Cells: Preclinical Development Jing Yang, M.D. Henry Klassen, M.D., Ph.D.

22 Donor Eligibility GTP tissue (including medical history) Pathology screening per FDA 21 CFR part 1271 Sample timing: within 24hrs of cell harvest/collect Hepatitis B Ag: ( ) Hepatitis C Ab: ( ) HIV 1&2 Ab: ( ) Syphilis: ( ) WNV: ( ) EBV Ab: ( ) CMV total Ab: (+) HSV IgG: (1:80) HSV IgM: ( ) hrpc from GTP tissue and eligible for cell therapy.

23 GLP/cGMP compatible cell culture hrpc cultured under GLP/cGMP compatible condition

24 Purity Cell isolation and cell culture Isolation 17~18 weeks Trypsinized cells No serum recovery Cell Culture Serum-free medium No antibiotics or antibiotics for first week followed by antibiotic-free medium for 6 weeks No anti-fungal agent Trypsin Express No trypsin inhibitor Avoiding occult contamination during cell preparation: No animal components involved! No bacterial or fungal contamination can hide in antibiotics/fungicidefree medium!

25 Identity Morphology: Live cell monitoring Day 0 (P0d0) Day 4 (P0d4) Day 7 (P1d2) Day 14 (P2d4) Day 19 (P3d5) Day 26 (P4d7) Day 30 (P5d4) Day 35 (P6d5) Day 41 (P7d6) Day 47 (P8d6) Day 51 (P9d4) Day 56 (P10d5) 200µm

26 Identity Growth Kinetics: jcell 1.0 Note: each dot is a passage. hrpcs have limited life time in vitro. They can proliferate up to 8w (passage 10), then stop dividing. The growth kinetics are reproducible.

27 Identity Immunocytochemistry Nestin/DAPI Vimentin/DAPI 95% 100% Sox2/DAPI 90% SSEA-1/DAPI 12% GD2/DAPI 6% Ki-67/DAPI 50% 100 µm

28 Identity B3-tubulin/DAPI 40% GFAP/DAPI 5% GDNF/DAPI 90% hrpc express Nestin, Vimentin, Sox2, SSEA- 1(CD15), GD2, Ki- 67,B3-tubulin, GFAP, GDNF biomarkers. 100 µm

29 Identity qpcr heap map Low expression FUT4 High expression Late passage Early passage

30 Identity Real time RT-PCR (time point gene profile) 5.0 d19 d35 d Fold change (Log 10 ) FUT4 Progenitor/Proliferation Phenotype Development Apoptosis Others Immune Control (fibroblast)=1 endogenous control: β-actin

31 Identity Microarray (RPC vs. BPC) PCA RPC vs. BPC 599 down-regulated genes 977 up-regulated genes RPC BPC Transcription factors Signaling molecules ECM cell adhesion

32 Identity Flow Cytometry

33 Identity Cell Viability Cell viability 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 99% 99% 98% 98% Before needle Cell Viability-27G Needle After needle 95% 95% 92% 92% 0 hr 1 hr 2.5 hr 3.5 hr Time point

34 Cytotoxicity study Steroid (Triamcinolone Acetonide) * Clinical dose Conclusion: Clinical dose of Triamcinolone Acetonide will NOT effect cell proliferation, but high dose (>10x clinical dose) will decrease the cell proliferation.

35 Medium optimization Advanced DMEM/F12 vs. DMEM/F12 * Conclusion: Advanced DMEM/F12 increase the yield 18-29%

36 Medium optimization Osmolarity Normal osmolarity: DMEM/F12 (300~318 mosm/kg) Low osmolarity: KnockOut DMEM/F12 (276 mosm/kg) exp_1 3 days exp_2 5 days * exp_3 3 days Conclusion: Low osmolarity will NOT increase the cell proliferation in either N2 or B27 supplemented medium.

37 Medium supplement optimization N2 vs. B27(XF) DMEM/F12 KnockOut DMEM/F12 Conclusion: B27 (XF) increase the yield, it could be candidate for hrpc culture in the future even though N2 is good, it needs more work to prove the cell therapy efficacy.

38 Medium optimization Ascorbic Acid Cell Viability (% control) 160% 140% 120% 100% 80% 60% 40% 20% 0% * * * * * contrl 0.025mg/ml 0.05mg/ml 0.1mg/ml 0.2mg/ml 0.4mg/ml Conclusion: VitC will increase yield ~30% Fresh VitC could be added every 2 days (daily added is not necessary) 0.1mg/ml (or 0.05mg/ml) will be the candidate conc (consistent with Yang previous paper in Stem Cells ).

39 Medium optimization Recombinant human albumin Advanced DMEM/F12 (SM) Basic DMEM/F12 Cell Viability (% control) 120% 100% 80% 60% 40% 20% * Cell Viability (% contrl) 140% 120% 100% 80% 60% 40% 20% * * * 0% contrl 0.04mg/ml 0.2mg/ml 1.0mg/ml 5.0mg/ml 25.0mg/ml albumin concentration 0% contrl 0.04mg/ml 0.2mg/ml 1.0mg/ml 5.0mg/ml albumin concentration Conclusion: Recombinant human albumin will NOT increase yield in Advanced SM Recombinant human albumin (1.0mg/ml) will increase yield up to 27% in Basic SM Ref: Albumin in VH is 1.0mg/ml

40 Low oxygen study P4 P5 P6 P7 P8 P9 P10 P3 P5 P6 P7 20% O2 3% O2

41 Low oxygen study WCB MCB MCB

42 Low oxygen study Working Cells Banks, jcell 2.0

43 jcell Preclinical Testing

44 Immune response study Flow Cytometry

45 Potency Multipotency (RA differentiation)

46 Sterility test Endotoxin Test Fungi Test Negative Negative Associates of Cape Cod, Inc (ACC): FDA approved, one of the world s largest manufacturers of products developed to detect bacterial endotoxin and fungi.

47 Sterility test Mycoplasma Test negative positive Positive Control Negative Control hrpc hrpc mycoplasma test: negative

48 Safety test Cytogenetic analysis G-banded Karyotyping FISH 46, XX. The result supports the presence of a normal karyotype. Normal FISH Signal Pattern for Chromosomes 7 and 19 Cell Line Genetics: FDA accepted

49 Safety test Telomerase activity hrpc from different donations, cultured under different conditions, both early and late passage show low telomerase activities. Telomerase transcriptase (htert) gene expression (qpcr): low expression

50 Safety test Tumorigenesis study: in vitro Positive Control- retinoblastoma Different donation & time in culture h-rpc #1-3w Number of colony: 234±18/well Negative Control- fibroblast #2-3w #3-3w Number of colony: 0/well Tumorigenic study shows negative colony formation of hrpc from different donations, both early and late passage. #4-3w #4-3m Number of colony: 0/well

51 Safety test Tumorigenesis study: in vivo 1. Our prior allogeneic studies: no tumors found in 66 mice, 21 pigs, 12 cats using species-specific RPCs. 2. Short term toxicity study (hrpcs): no tumors found in 110 RCS rats. 3. Long term NOD/SCID gamma mice, toxicity study in progress. Plans: Biodistribution (qpcr): 10 organs Tumorigenisis/Pathology: eyes STEM 121 GFAP Isolectin B4

52 Stability/reproducibility Freeze-Thaw Study Storage: Liquid Nitrogen>2 years Shipping: living cells or frozen vials Normoxia hrpc (jcell 1.0)

53 jcell Clinical Safety Trial China jcell Patients with end stage RP Open label Single intraocular injection Topical anesthesia No immune suppression

54 Characteristics of Study Eyes Patient No. Age (yrs) Gender Diagnosis Eye/Lens Female RP L/Cataract Female RP R/IOL Male RP L/IOL All patients legally blind

55 Slit lamp/30d lens 001-day1 002-day1 003-day1 002-day2

56 B Scan 001-Initial months

57 Fundus Photos 002-Initial 002-day3

58 Intraocular Pressures of Study Eyes Patient No. Intraocular Pressure (mmhg) Initial Final 001 Normal Normal 002 Normal Normal 003 Normal Normal

59 #002 #001 #003 Increase 20 letters Cataract NLP

60 Patient /200 20/100 20/70 20/50 20/40 20/30 20/25 20/20 Increase 20 letters

61 Clinical Trail: Summary Vision improved in all patients (one patient = 20 letters) No surgical complications No immune rejection without immune suppression or tissue typing No detectible cell proliferation in vivo based on all examinations (slitlamp, 30D lens, B scan, fundus photos, etc.) No tumor formation up to 27 months (>2yrs)

62 Targeted Diseases Retinal degenerations Retinitis Pigmentosa (RP), other dystrophies Age-Related Macular Degeneration (AMD) Retinal detachment Other retinal diseases Gavin Herbert Eye Institute