Vision Research Symposium Conference Presentation
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"The use of embryonic stem cells in regenerative medicine" 12/7/206
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Vision Research Symposium Conference Presentation
- 1. The use of embryonic stem cells in regenerative medicine Robert Lanza, MD VP Research & Scientific Development Advanced Cell Technology and Adjunct Professor Wake Forest University School of Medicine
- 2. Alzheimer’s Dwarfism Parkinson’s Strokes Epilepsy Hemophilia Kidney failure Chronic pain Cancer Infertility Burns AIDS Muscular dystrophy ALS Affective disorders Macular degeneration Hypoparathyroidism Heart disease Liver failure Enzymatic defects Diabetes Osteoarthritis Multiple sclerosis Huntington’s Hypocholesterolemia Rheumatoid arthritis Atherosclerosis Ulcers Spinal cord injuries
- 6. Anatomy & Function of RPE • Immune barrier • Absorption of stray light • Vit A metabolism & transport • Phagocytosis of shed photoreceptor segments FUNCTIONS OF RPE
- 7. Diseases Associated with RPE dysfunction • Age-related macular degeneration • Retinitis pigmentosa • Stargardt's disease • Best's vitelliform macular dystrophy • Leber's congenital amaurosis
- 8. Animal models of RPE dysfunction • Royal College of Surgeons (RCS) rat (MERTK mutation, phagocytosis-impaired) • Mouse models: RPE65 -/-; rd-mouse (cGMP- phosphodiesterase mutation, loss of rods) • Dog (Briard, RPE65 mutation) • Monkey (rhesus monkey with naturally occurring macular degeneration)
- 9. Transplantation of RPE in Humans Associated problemsSources of RPE cells * autologous tissue * cell lines * donor tissue (adult, fetal) safety ethical Batch-to-batch variation may have impaired function limited supply Potential tumorigenicity
- 10. Advantages of ECS-derived Tissues for Regenerative Medicine • Unlimited supply • Can be derived under GMP conditions pathogen- free • Can be produced with minimal batch to batch variation • Can be thoroughly characterized to ensure optimal performance
- 12. [All hES cell lines studied reproducibly generated RPE lines that could be passaged, characterized, and expanded] •WiCell hES cell lines (23 RPE lines generated) WA01 WA09 WA07 •Harvard hES cell lines (22 RPE lines generated) HUES1 HUES6 HUES2 HUES7 HUES3 HUES8 HUES5 HUES10 •ACT hES cell lines (25 RPE lines generated) MA01 MA03 MAJ1 MA04 MA09 MA14 MA40 RPE can be generated from hES cells
- 13. x400 x200 hES-RPE express RPE markers (bestrophin & CRALBP) -- 32 -- 46 -- 78 CRALBP Mw a b c bestrophin bestrophin CRALBP Immunostainin g Western blot
- 14. PEDF RPE65 RT-PCR 1 2 1 2 1 – fetal RPE 2 – hES-RPE hES-RPE express RPE65 and PEDF
- 15. DB X15,200 x7000 Phagocytosis of latex beads (electron microscopy) Latex beads pigment
- 16. Stages of RPE isolation from spontaneously differentiating hES cells 35 mm plate one of the clustersone of the clusters cell suspension at plating 4 days x100x200 x200 7 days x200 Passage 1 -- 25 daysPassage 1 -- 25 days x200 x0.75
- 17. hES-RPE vs. its in vivo counterpart RPE hES-RPE cobblestone, pigmented transdifferentiation-differentiation phagocytosis molecular markers RPE65 CRALBP bestrophin PEDF MERTK
- 18. Gene expression profiling of hES-RPE vs. their in vivo counterpart
- 19. RPE in animal studies
- 20. RPE transplantation into subretinal space of RCS rats (in collaboration with Raymond Lund, University of Utah) RCS rats naturally become blind in several weeks due to RPE degeneration and photoreceptor death Study design cell line RPE (H9) Control: culture medium Tests: head tracking (behavior) electroretinogram (ERG) histology In vitro assessment: molecular markers of RPE morphology and behavior
- 21. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 R PE65 bestrophin C R A LB P PED F Pax6 G A PD H H9 RPE used for transplantation in RCS rats
- 22. cone hES-RPE ERG at P60 Amplitude(uV) 40 a-hES-RPE a-sham b-hES-RPE b-sham cone b-sham 180 0 20 60 80 120 140 160 100 hES-RPE transplantation into subretinal space of RCS rats Optomotor at P100 hES-RPE Sham Untreated 0.5 0.3 0.4 0.2 0 0.1 Relativeacuity(c/d)
- 25. Summary • hES-RPE is similar to its in vivo counterpart by multiple parameters (morphology, behavior, phagocytosis, molecular markers) • hES-RPE can be reproducibly generated from hES cells • hES-RPE attenuates photoreceptor loss in animal model of retinal degeneration hES-RPE advantages • Minimize batch-to-batch variation • Can be derived under GMP conditions • Can be produced from feeder-free hES cells • Can be easily generated in large quantities (for pathogen & safety assessment, and pre-clinical & clinical studies)
- 26. hES-RPE: ongoing pre-clinical studies and research goals • functional studies in animal models with different batches of cells (more and less differentiated, different passages, different lines) • finding reliable markers for predicting therapeutic value of newly generated cells • studies of hES-RPE survival on Bruch’s membrane • production of hES-RPE under GMP conditions • dosage and safety studies of hES-RPE in animal models
- 29. Generation of ES cells using parthenogenesis
- 31. WBC colony from cloned stem cells
- 32. •Cardiovascular disease costs the US $329 billion annually
- 34. Is it possible to generate ES cells without destroying embryos? • The most basic objection to ES cell research is that it deprives embryos of any further potential to develop into complete human beings • For a decade, PGD has been used successfully to remove a single cell (blastomere) for genetic testing without interfering with the developmental potential of the biopsied embryo. Over 2,000 healthy babies have been born using this procedure • Question: Can such a biopsied cell be used to generate ES cells?
- 36. Biopsy Procedure Live Young Biopsied 23/47 (49%) Non-Biopsied 38/75 (51%)
- 38. Oct-4 Alk Phos Oct-4 Alk Phos SSEA-1 Troma-1 Laz-Z Lac-Z b III tubulin Ectoderm Smooth muscle actin Endoderm alpha feto-protein Endoderm
- 40. Biopsy Procedure (Human Embryo) Blastocyst Biopsied 6/8 (75%) Non-Biopsied 1/4 (75%)
- 41. Derivation of hES Cells From Single Blastomeres Blastomere biopsy GFP hESs Feeders Feeders 1 or 2 single blastomeres biopsied and co-cultured with parent embryo Multiple single blastomeres biopsied and co-cultured together
- 42. Blastomere divided outgrowth first passage second passage established line Stages of Derivation of hES Cells From Single Blastomere
- 43. Markers of Pluripotency Alkaline phosphatase TRA I-81 SSEA-4 TRA 1-60Oct-4 SSEA-3
- 44. Endoderm - Cdx2 (intestine)Ectoderm – nestin (neural tissue) Mesoderm - smooth muscle actin Kidney tissue teratoma Teratoma Formation in NOD-SCID Mice
- 45. In Vitro Differentiation Into Cells of Specific Therapeutic Interest RPECapillary structures Ac-LDL Bestroph in MA01 (blastomere-derived hESC line) generated hematopoietic progenitors 5-10 times more efficiently than H9 and 3-5 times more efficiently than H1 Ac-LDL MA09 (blastomere-derived hESC line) generated vascular/endothelial progenitors 1- 2 times more efficiently than H1 and H9 MA01 & MA09 (blastomere-derived hESC lines) generated neural progenitors without the need for EB-intermediates, stromal feeder layers, or low-density passaging
- 46. MA01 MA09 karyotypeCharacterization of Single Blastomere-Derived hES Cell Lines
- 47. Markers H1 MA01 MA09 Markers H1 MA01 MA09 No Presence of Y Chromosome or GFP Gene Setected by PCR X YGFP
- 48. FES primerpair WA31 primer pair ds526 216 220 224 228 142 151 154 192 196 200 204 230 238 242 250 H1 1 H9 2 ACT 4 3 MA01 4 MA09 5 MA04 7 BLANK 8 ds592 ds417 170 178 182 186 190 173 177 181 H1 1 H9 2 ACT 4 3 MA01 4 MA09 5 MA04 7 BLANK 8 Checkerboard Fingerprints H1 H1 MA01 MA09 MA09 MA01