NAS Conference Presentation


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"Embryonic Stem Cell Lines Derived From Single Blastomeres (and Altered Nuclear Transfer)"


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NAS Conference Presentation

  1. 1. Embryonic Stem Cell Lines Derived From Single Blastomeres(and Altered Nuclear Transfer)Robert Lanza, MDVP Research & Scientific DevelopmentAdvanced Cell Technologyand Adjunct ProfessorWake Forest University School of Medicine
  2. 2. Is it possible to generate ES cells without destroying embryos?• The most basic objection to ES cell research is that it deprives embryosof 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 developmentalpotential of the biopsied embryo. Over 2,000 healthy babies have beenborn using this procedure• Question: Can such a biopsied cell be used to generate ES cells?
  3. 3. Biopsy ProcedureLive YoungBiopsied 23/47 (49%)Non-Biopsied 38/75 (51%)
  4. 4. Oct-4Alk PhosOct-4Alk PhosSSEA-1 Troma-1Laz-Z Lac-Zb III tubulinEctodermSmooth muscle actinEndodermalpha feto-proteinEndoderm
  5. 5. Biopsy Procedure (Human Embryo)BlastocystBiopsied 6/8 (75%)Non-Biopsied 1/4 (75%)
  6. 6. 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
  7. 7. Derivation of hESCs Without Embryo DestructionStep 1: Biopsied embryo & blastomeres develop independentlyStep 2: Blastomere co-culturedwith hESC and ES-cells formedBiopsied Embryo MA61 --hES cellsBiopsied Embryo MA61 –Hatching blastocyst
  8. 8. Blastomere divided outgrowth first passagesecond passage established lineStages of Derivation of hES Cells From Single Blastomere
  9. 9. Markers of PluripotencyAlkaline phosphataseTRA I-81 SSEA-4TRA 1-60Oct-4SSEA-3
  10. 10. RT-PCR Analysis/Expression of Markers of PluripotencyOct-4NanogGAPDHLane 1: no templateLane 2: negative controlLane 3: MAO1Lane 4: MA09Lane 5: H1
  11. 11. In vitro differentiationCharacterization of Single Blastomere-Derived hES Cell Linesββββ III tubulinectodermSmooth muscle actinendodermalpha feto-proteinendoderm
  12. 12. Endoderm - Cdx2 (intestine)Ectoderm – nestin (neural tissue) Mesoderm - smooth muscle actinKidney tissueteratomaTeratoma Formation in NOD-SCID Mice
  13. 13. In Vitro Differentiation Into Cells of Specific Therapeutic InterestRPECapillary structuresAc-LDLBestrophinMA01 (blastomere-derived hESC line) generated hematopoietic progenitors 5-10 times moreefficiently than H9 and 3-5 times more efficiently than H1Ac-LDLMA09 (blastomere-derived hESC line) generated vascular/endothelial progenitors 1-2 timesmore efficiently than H1 and H9MA01 & MA09 (blastomere-derived hESC lines) generated neural progenitors without the need forEB-intermediates, stromal feeder layers, or low-density passaging
  14. 14. MA01 MA09karyotypeCharacterization of Single Blastomere-Derived hES Cell Lines
  15. 15. MarkersH1MA01MA09MarkersH1MA01MA09No Presence of Y Chromosome or GFP Gene Setected by PCRXYGFP
  16. 16. DAx 8.0 4/10/2006 2:19:35PM mike700 750 800Time (s)0100002000030000YDAx 8.0 4/10/2006 2:22:03PM mike700 750Time (s)01000020000YMA01 MA09DAx 8.0 4/10/2006 2:03:50PM mike700 750 800Time (s)01000020000YH1Characterization of Single Blastomere-Derived hES Cell LinesMicrosatellite analysis confirming unique identity of the cell lines
  17. 17. 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
  18. 18. Altered Nuclear Transfer (ANT)• “Methods of Producing Differentiated Progenitor Cells and Lineage-detective ES Cells”(Idea/patent filings 1999/2000 on… ACT Patent NZ518191, WO Patent No. 0129206…)• Presented Dec 2004 at the President’s Council on Bioethics by Council Member William Hurlbut– Outlined in White Paper: “Alternative Sources of Human Pluripotent Stem Cells” May 2005– Conceptually based on SCNT– Genetically alter somatic cell before transferred to enucleated egg, such that the resultingbiological entity lacks the essential capacities of a human embryo– “Such an entity would be a ‘Biological Artifact’ – “ethically equivalent to tissue culture,teratoma, or mole.”– Derivation of hES cells from the developing entity “would not be killing or harming, for thereis no living being here to be killed or harmed.”• ANT has garnered broad support from the pro-life community and from some influential membersof the US Congress. Endorsed by prominent Catholic moral theologians & ethicists. RomanCatholic Archbishop of San Francisco, William Levada, wrote to President Bush assuring him ofhis support for ANT.
  19. 19. Generation of hESCs From Cdx2-deficient BlastocystsGeneration of hESCs
  20. 20. “Choose your method of destruction”
  21. 21. A Few Questions to Consider• Is this “artifact” just a bundle of cells or a defective embryo?• Should science use cloning and genetic manipulation to deliberatelycreate crippled human embryos?• Addressing ethical concerns is important, but should we carry outsuch manipulations –not for medical or scientific reasons – but ratherto address theological problems?• Will ANT appease the pro-life and Catholic communities and helpmove hESC therapies into the clinic? Or rather, is it a disingenuousattempt to frustrate and oppose current avenues of hESC research?
  22. 22. Acknowledgements• Irina Klimanskaya• Young Chung• Sandy BeckerShi-Jiang LuTong LiSadhana Agarwal