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Induced Pluripotent Stem Cells ( iPSCs ): Frontiers in Reprogramming

Induced Pluripotent Stem Cells ( iPSCs ): Frontiers in Reprogramming. Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany.

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Induced Pluripotent Stem Cells ( iPSCs ): Frontiers in Reprogramming

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  1. Induced Pluripotent Stem Cells (iPSCs): Frontiers in Reprogramming Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany

  2. Epigenetic States Alter Developmental Pathways and Can Be Used to Modify Developmental State Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

  3. Methods of Factor Delivery Nimet Maherali and Konrad Hochedlinger; Cell Stem Cell 3, December 4, 2008 Presentation title in footer | 00 Month 0000

  4. ES-Cell Transcription Factors and Their Role in Reprogramming Activation of pluripotency regulators Repression function Activation of metabolic and proliferative programs Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

  5. Role of 4Factors During Reprogramming Rupa Sridharan et al.(2009) :Cell, Volume 136, Issue 2, Pages 364-377 Presentation title in footer | 00 Month 0000

  6. The Process of Generation of Induced Pluripotent Cells (iPS) Hochedlinger,K and Plath, K. Development 136, 509-523 (2009) Presentation title in footer | 00 Month 0000

  7. Methods of iPS Generation: Small Molecules Presentation title in footer | 00 Month 0000

  8. Reprogramming Process Takashi Tada (2008); Cell Stem Cell, Volume 3, Issue 2,Pages 121-122 Presentation title in footer | 00 Month 0000

  9. All Mycs Are Not Created Equal!! •c-Myc, L-Myc, and N-Myc all have the ability to contribute to iPSC generation •c-Myc promotes iPS cell generation, but has significant transformative ability, as well; L-Myc, by contrast, shows very low transformative ability Nakagawa et al. PNAS 107(32): Presentation title in footer | 00 Month 0000

  10. Assessment of iPS Potency • Morphology ID • Unlimited self-renewal; • Visual ID • Molecular ID • Protein level expression of key pluripotency factors (Oct 4, Nanog) and key specific surface antigens (SSEA-4, Tra-1-60/-81) • Functional Telomerase expression • Expression of genes involved in retroviral silencing (de novo methyltransferases and Trim28/ transgene independence • Epigenetic similarity of ESCs including demethylation at the promoters of pluripotency genes, X chromosome reactivation • Bivalent domains of developmental genes consisting of overlapping histone modifications • Histone 3 K4 trimethylation/unmethylated Histone 3 K27 = hallmark of iPS and ES cells • Histone 3 K4 umethylated/trimethylated Histone 3 K27 =hallmark of differentiated cells • Histone 3 K4 trimtehylation + Histone 3 K27 trimethlyation = hallmark of partially reprogrammed iPS cells • Functional ID: (Gold Standards) • Mouse: Tetraploid complementation assay (fused blastocyte+iPS cell gives rise to live pup) • Human: Teratoma formation Presentation title in footer | 00 Month 0000

  11. STEMCCA: Single Vector Delivery of 4 Transcription Factors TetOn/TetOff Inducible Promoter Constitutive Promoter/loxP-Flanked Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74. Presentation title in footer | 00 Month 0000

  12. Human Reprogramming Requires Longer Time Mouse Human 12 Presentation title in footer | 00 Month 0000 Slower reprogramming compared to mouse • Picking colonies: 18-25 vs. 10-12 days • Mouse iPS can be manipulated similarly as ESC after first passage • During the first 3 passages, human iPS clones require longer length of time to grow (~18-25 days at p0 and 10-12 days for p1-p3 each passage) to sufficient size for passaging. Approx: 50-60 days to establish

  13. Time Course of Human iPS Colony Formation Timing: Infection to colony formation (p0): 18-25 days p0 to p3: 10-12 days for each passage; 50-60 days total p3 to p4: 7 days 13 Presentation title in footer | 00 Month 0000

  14. Removal of c-myc Requires Higher MOI and Displays Slower Reprogramming Kinetics • Fewer clones (4 vs. 7 – 15) • Higher MOI required (100 vs. 20) • Slower kinetics (24 vs. 14 days) 14 Presentation title in footer | 00 Month 0000

  15. OKS/L-Myc STEMCCA: The Latest Polycistronic Vector for hiPSC Generation •Human forms of Oct-4, Klf4, and Sox2 •L-Myc in place of c-Myc •loxP-flanked, so can be removed with Cre recombinase Presentation title in footer | 00 Month 0000

  16. Timecourse of OKS/L-Myc Reprogramming Presentation title in footer | 00 Month 0000

  17. hiPSC Boost II Presentation title in footer | 00 Month 0000

  18. Oh, The Difference Small Molecules Can Make!! SSEA-4 TRA-1-60 SSEA-4 TRA-1-60 Presentation title in footer | 00 Month 0000

  19. Healthier, More Robust iPS Production Against Different Backgrounds mTeSR without treatment mTeSR with treatment StemPro without treatment StemPro with treatment Presentation title in footer | 00 Month 0000

  20. iPSC Colonies Generated with OKS/L-Myc and iPS Boost II Presentation title in footer | 00 Month 0000

  21. Induction of Sox-2 in Fibroblasts from a GFP-Sox-2 Transgenic Mouse Model GFP-Sox-2 transgenic animals Fibroblasts harvested and cultured Cultures transduced with STEMCCA Cells assayed via FACS against GFP expression Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Presentation title in footer | 00 Month 0000

  22. Expression of Pluripotent Markers in STEMCCA-Transduced Cells Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Presentation title in footer | 00 Month 0000

  23. Following Excision of loxP-Flanked Vector, iPSCs Undergo Directed Differentiation Without excision, STEMCCA is reinducible 1. Chimeric embryos generated with STEMCCA-generated iPS cells 2. MEFs generated 3. TetO promoter activated by doxycycline addition to cell culture Sommer, C.A.; et al. 2009. Stem Cells 27(3): 543-549. Sommer, C.A.; et al. 2010. Stem Cells 28(1): 64-74. Presentation title in footer | 00 Month 0000

  24. SSEA-4 and TRA-1-60 As Stage-Specific Markers of Reprogramming p3 non-iPS cells At this stage, can use Human iPS Selection Kit to exclude SSEA-4 negative colonies and focus on SSEA-4 positive colonies. 24 Presentation title in footer | 00 Month 0000

  25. Live Staining to Characterize Fully Reprogrammed hiPS Cells Live stain without having to sacrifice colony for analysis -30 min for SSEA4 and Tra-1-60 20 min for Hoechst 50 minutes total Hoechst Dim SSEA-4+ Tra1-60+ Antibodies can be washed off without altering morphology and proliferation 25 Presentation title in footer | 00 Month 0000

  26. Michael L. Moeller, MS, PhD Field Application Scientist III Bioscience Division EMD Millipore A Division of Merck KGaA Darmstadt Germany michael.moeller@merckgroup.com

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