Vision Research Symposium Conference Presentation


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"The use of embryonic stem cells in regenerative medicine"


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Vision Research Symposium Conference Presentation

  1. 1. The use of embryonic stem cells in regenerative medicineRobert Lanza, MDVP Research & Scientific DevelopmentAdvanced Cell Technologyand Adjunct ProfessorWake Forest University School of Medicine
  2. 2. Alzheimer’sDwarfismParkinson’sStrokesEpilepsyHemophiliaKidney failureChronic painCancerInfertilityBurnsAIDSMuscular dystrophyALSAffective disordersMacular degenerationHypoparathyroidismHeart diseaseLiver failureEnzymatic defectsDiabetesOsteoarthritisMultiple sclerosisHuntington’sHypocholesterolemiaRheumatoidarthritisAtherosclerosisUlcersSpinal cordinjuries
  3. 3. 10203040506070809010019881989199019911992199319941995199619971998199920002001YearNumberofPatients(inthousands)Waiting ListOrgans Transplanted
  4. 4. Anatomy & Function of RPE• Immune barrier• Absorption of stray light• Vit A metabolism & transport• Phagocytosis of shedphotoreceptor segmentsFUNCTIONS OF RPE
  5. 5. Diseases Associated with RPE dysfunction• Age-related macular degeneration• Retinitis pigmentosa• Stargardts disease• Bests vitelliform macular dystrophy• Lebers congenital amaurosis
  6. 6. Animal models of RPEdysfunction• 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 maculardegeneration)
  7. 7. Transplantation of RPE in HumansAssociated problemsSources of RPE cells* autologous tissue* cell lines* donor tissue (adult, fetal) safety ethical Batch-to-batchvariationmay have impaired function limited supplyPotential tumorigenicity
  8. 8. Advantages of ECS-derived Tissues for RegenerativeMedicine• Unlimited supply• Can be derived under GMP conditions pathogen-free• Can be produced with minimal batch to batchvariation• Can be thoroughly characterized to ensure optimalperformance
  9. 9. [All hES cell lines studied reproducibly generated RPE linesthat could be passaged, characterized, and expanded]•WiCell hES cell lines (23 RPE lines generated)WA01 WA09WA07•Harvard hES cell lines (22 RPE lines generated)HUES1 HUES6HUES2 HUES7HUES3 HUES8HUES5 HUES10•ACT hES cell lines (25 RPE lines generated)MA01 MA03 MAJ1MA04 MA09MA14 MA40RPE can be generated from hES cells
  10. 10. x400x200hES-RPE express RPE markers (bestrophin &CRALBP)-- 32-- 46-- 78CRALBPMwa b cbestrophinbestrophinCRALBPImmunostainingWestern blot
  11. 11. PEDF RPE65RT-PCR1 2 1 21 – fetal RPE2 – hES-RPEhES-RPE express RPE65 and PEDF
  12. 12. DBX15,200x7000Phagocytosis of latex beads (electron microscopy)Latex beadspigment
  13. 13. Stages of RPE isolation from spontaneously differentiating hES cells35 mm plate one of the clustersone of the clusters cell suspension at plating4 daysx100x200x2007 daysx200Passage 1 -- 25 daysPassage 1 -- 25 daysx200x0.75
  14. 14. hES-RPE vs. its in vivo counterpartRPE hES-RPEcobblestone, pigmentedtransdifferentiation-differentiationphagocytosismolecular markersRPE65CRALBPbestrophinPEDFMERTK
  15. 15. Gene expression profiling of hES-RPE vs. their in vivocounterpart
  16. 16. RPE in animal studies
  17. 17. RPE transplantation into subretinal space of RCS rats(in collaboration with Raymond Lund, University of Utah)RCS rats naturally become blind in several weeksdue to RPE degeneration and photoreceptor deathStudy designcell line RPE (H9)Control: culture mediumTests:head tracking (behavior)electroretinogram (ERG)histologyIn vitro assessment:molecular markers of RPEmorphology and behavior
  18. 18. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3RPE65bestrophinCRALBPPEDFPax6GAPDHH9 RPE used for transplantation in RCS rats
  19. 19. conehES-RPEERG at P60Amplitude(uV)40a-hES-RPE a-sham b-hES-RPE b-sham coneb-sham1800206080120140160100hES-RPE transplantation into subretinal space of RCS ratsOptomotor at P100hES-RPE Sham Untreated0.
  20. 20. 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 degenerationhES-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)
  21. 21. hES-RPE: ongoing pre-clinical studiesand 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 valueof 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
  22. 22. Generation of ES cells using parthenogenesis
  23. 23. WBC colony from cloned stem cells
  24. 24. •Cardiovascular disease costs the US $329 billion annually
  25. 25. Is it possible to generate ES cells without destroyingembryos?• The most basic objection to ES cell research is that it deprivesembryosof any further potential to develop into complete human beings• For a decade, PGD has been used successfully to remove a singlecell (blastomere) for genetic testing without interfering with thedevelopmental potential of the biopsied embryo. Over 2,000healthy babies have been born using this procedure• Question: Can such a biopsied cell be used to generate ES cells?
  26. 26. Biopsy ProcedureLive YoungBiopsied 23/47 (49%)Non-Biopsied 38/75 (51%)
  27. 27. Oct-4Alk PhosOct-4Alk PhosSSEA-1 Troma-1Laz-Z Lac-Zb III tubulinEctodermSmooth muscle actinEndodermalpha feto-proteinEndoderm
  28. 28. Biopsy Procedure (Human Embryo)BlastocystBiopsied 6/8 (75%)Non-Biopsied 1/4 (75%)
  29. 29. Derivation of hES Cells From Single BlastomeresBlastomere biopsyGFP hESsFeedersFeeders1 or 2 single blastomeres biopsiedand co-cultured with parent embryoMultiple single blastomeresbiopsied and co-cultured together
  30. 30. Blastomere divided outgrowth first passagesecond passage established lineStages of Derivation of hES Cells From Single Blastomere
  31. 31. Markers of PluripotencyAlkaline phosphataseTRA I-81 SSEA-4TRA 1-60Oct-4SSEA-3
  32. 32. Endoderm - Cdx2 (intestine)Ectoderm – nestin (neural tissue) Mesoderm - smooth muscle actinKidney tissueteratomaTeratoma Formation in NOD-SCID Mice
  33. 33. In Vitro Differentiation Into Cells of Specific Therapeutic InterestRPECapillary structuresAc-LDLBestrophinMA01 (blastomere-derived hESC line) generated hematopoietic progenitors 5-10 timesmore efficiently than H9 and 3-5 times more efficiently than H1Ac-LDLMA09 (blastomere-derived hESC line) generated vascular/endothelial progenitors 1-2 times more efficiently than H1 and H9MA01 & MA09 (blastomere-derived hESC lines) generated neural progenitors without theneed forEB-intermediates, stromal feeder layers, or low-density passaging
  34. 34. MA01 MA09karyotypeCharacterization of Single Blastomere-Derived hES Cell Lines
  35. 35. MarkersH1MA01MA09MarkersH1MA01MA09No Presence of Y Chromosome or GFP Gene Setected by PCRXYGFP
  36. 36. FES primerpair WA31 primer pair ds526216 220 224 228 142 151 154 192 196 200 204 230 238 242 250H1 1H9 2ACT 4 3MA01 4MA09 5MA04 7BLANK 8ds592 ds417170 178 182 186 190 173 177 181H1 1H9 2ACT 4 3MA01 4MA09 5MA04 7BLANK 8Checkerboard FingerprintsH1H1MA01MA09MA09MA01