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Konrad Hochedlinger

Cmcwintersymposium’12. Induced Pluripotent Stem Cells (iPSCs). Konrad Hochedlinger Department of Stem Cell and Regenerative Biology Howard Hughes Medical Institute Harvard Stem Cell Institute. Career. B.S. Biology University of Vienna, Austria. 1997. M.S. Genetics

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Konrad Hochedlinger

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  1. Cmcwintersymposium’12 Induced Pluripotent Stem Cells (iPSCs) Konrad Hochedlinger Department of Stem Cell and Regenerative BiologyHoward Hughes Medical InstituteHarvard Stem Cell Institute

  2. Career B.S. Biology University of Vienna, Austria. 1997 M.S. Genetics Research Institute of Molecular Pathology, Vienna, Austria. 1999 Ph.D. Mammalian Development, Research Institute of Molecular Pathology, Vienna, Austria. Erwin Wagner 2000-2003 Post Doc. 2004-2006 Principal Investigator, Department of Stem Cell & Regenerative BiologyHoward Hughes Medical InstituteHarvard Stem Cell Institute, Boston. Rudolf Jaenisch 2007-present

  3. Awards • Genzyme Postdoctoral Fellowship at Whitehead Institute, 2004. • Scholar Award, Sydney Kimmel Foundation for Cancer Research, 2007. • NIH Director’s Innovator Award, 2007. • MIT Technology Review’s Top 35 Innovators Under 35, 2008. • Howard Hughes Medical Institute Early Career Scientist Award, 2009. • ISSCR Outstanding Young Investigator Award, 2009.

  4. Area of Research • Molecular mechanisms underlying pluripotency and nuclear reprogramming. • Transgenic and knock-out mice. • Manipulated murine and human ES cells. • Genome-wide approaches including RNAi and chemical screening. • Generate custom-tailored cells for treating and understanding disease.

  5. Pluripotent Stem cells Cells that exhibit: • Pluripotency: capacity of a single cell to generate all cell lineages of the developing and adult organism • Self renewal: ability of a cell to proliferate in the same state. Types of pluripotent stem cells: • Embryonic stem cells. • Embryonic carcinoma cells. • Embryonic germ cells. • Epiblast stem cells. • Induced pluripotent stem cells.

  6. Somatic Cell Nuclear Reprogramming Dolly (05.07.1996 –14.02.2003) Ian Wilmut, Roslin Institute Nuclear reprogramming to a pluripotent state by three approaches; Shinya Yamanaka & Helen M. Blau; Nature|Vol 465|10 June 2010|doi:10.1038/nature09229.

  7. Dr. ShinyaYamanaka, PhD Dr. Kazutoshi Takahashi, PhD Human iPSCs

  8. Reprogramming Factors – Magic Four 24 candidates expressed in embryonic stem cells 10 candidates 4 candidates Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Takahashi and Yamanaka. Cell. 126, 663-676, 2006.

  9. iPSCs has been generated from Mouse (Yamanaka et al., 2006) Humans (Yamanaka et al., 2007) Rhesus monkey (Liu et al., 2008) Rats(Liao et al., 2009; Li et al., 2009) Canine (Shimada, H. et al, 2010) Porcine (Esteban, M. A. et al., 2009) Marmoset (Wu, Y. et al., 2010) Rabbit (Honda, A. et al., 2010) Equine (Kristina Nagy et al., 2011 ) Avian (Lu et al., 2011)

  10. iPSCs – Clinical Applications • Ability to differentiate into many cell types. • Vastly renewable. • - Easily accessible. • - Individual-specific i.e. personalized or non-immunogenic.

  11. Reprogramming Menu Progress toward the clinical application of patient-specific pluripotent stem cells. Evangelos Kiskinis and Kevin Eggan. The Journal of Clinical Investigation. 2010

  12. Molecular events in iPS Generation Stochastic event Multiple barriers • Low Efficiency • Slow Kinetics Epigenetic Mechanisms that Regulate Cell Identity. Belmonte et al. Cell Stem Cell. Nov 2010. Pluripotency: Toward a gold standard for human ES and iPS cells. Journal of Cellular Physiology. July 2009.

  13. Molecular events in iPS Generation Pathway classification of the target genes of Yamanka factors in iPS cells using KOBAS. Fibroblast Reprogramming into iPSCs entails a Mesenchymal-to-Epithelial Transition Jinyan Huang et al., 2009; Hochedlinger, 2010.

  14. iPSCs – Clinical Applications Culture dish Clinic Obstacles in therapeutic application of iPSCs in humans (i) Use of harmful oncogenes as part of the reprogramming factors . (ii) Use of viral vectorsfor gene delivery that carry the risk of insertionalmutagenesis. (iii) Low efficiencyand slow kineticsof reprogramming. (iv) Lack of robust and reliabledifferentiation protocols for human iPS cells.

  15. Current Research Areas Efficiency and Kinetics Safety Disease Modeling Patient Care Methods for making induced pluripotent stem cells: reprogramming à la carte. Belmonte et al., 2011. Nature Review Genetics.

  16. Konrad’s contributions to iPSC research • Secondary reprogramming system. • Differentiation state of starting cells. • Endogenous level of reprogramming factors. Efficiency and Kinetics • Disease specific iPS. • Differentiation bias due to epigenetic memory. • Ease in gene targeting in hiPS with murine ES cell state. Disease Modeling • iPSC without viral integration. • Selection of bonafideiPS clone based on Imprinting pattern. Safety

  17. Metastable Human IPSCs with Mouse ESCs Property Schematic Representation of the strategy Colony Morphology FACS ANALYSIS

  18. LIF RESPONSIVENESS OF HLR5 CELLS Immunostaining Western blot analysis Gene expression analysis Flow Cytometry analysis Colony morphology Flow Cytometry analysis

  19. The hLR5 State Depends on Ectopic Pluripotency Factors but Is Poised for Reactivation of Endogenous Pluripotency Genes Colony morphology ChIP-qPCR analysis Q PCR analysis DNA methylation analysis Schematic representation of the generation of hLR5 cells

  20. Conversion of hLR5 Cells to a Stable Pluripotent State Schematic representation Karyotype Analysis LD-hIPS colony morphology Q PCR analysis Immunostaining Unbiased cluster analysis Scatter plots of microarray data H&E staining of teratomas Immunostaining

  21. hLR5 Cells Facilitate Transgenesis and Gene Targeting in Human Stem Cells Schematic representation of the human HPRT locus and the targeting construct Confirmation of functional knockout of the HPRT gene in targeted hLR5 cells PCR detection

  22. Application of the Intermediate hLR5 Cells

  23. Musheer Aalam JRF, Lab-2, CSCR

  24. Induction of iPSCs from Isogenic Chimeric Mice Clones between Passage 4 and 6 were analyzed.

  25. iPSCs retain transcriptional memory of somatic cell of origin Gra vs Gra iPSCs SMP vs SMP iPSCs Gra iPSCs vs SMP iPSCs

  26. iPSCs with same cell of origin cluster together Gra iPSCs Vs SMP iPSCs - 1,388 genes were differentially expressed. B iPSCs Vs TTF iPSCs -1,090 genes were differentially expressed. Global transcriptional profiling distinguishes iPSCs obtained from different genetically identical somatic cell types.

  27. Contribution Experimental variables on Gene Expression patterns Gra iPSCs Chi1 Vs Gra iPSCs Chi 2 Hierarchical clustering separated Gra iPSCs according to their origin form different animals. Differences due to cell of origin were stronger than those arising from variations in experimental conditions or animals.

  28. Correlation of gene expression patterns with epigenetic marks Genome wide restriction enzyme based methylation pattern analysis by“HpaII tiny fragment Enrichment by Ligation PCR” (HELP) assay. Unsupervised Hierarchical clustering analysis separated iPSCs into different groups according to cell of their origin No difference could be observed in promoter methylation pattern between Gra iPSCs and SMP iPSCs.

  29. ChIP for Histone Modifications

  30. iPSCs derived from different cell types have distinctive in vitro differentiation potential

  31. Continuous passaging of iPSCs abrogates Transcriptional, Epigenetic and Functional Difference Total no:of differentially expressed genes between various pairs were reduced from ~500-2000 in early passage culture to ~50 or even 0 in late passage iPSCs.

  32. Methylation analysis- HELP assay of Late passage iPSCs In Vitro Differentaiation of Late passage iPSCs

  33. Discussion • Complete reprogramming is a gradual process that continues beyond the acquisition of a bonafideiPSC state. • Two possible mechanisms for observed loss of epigenetic and transcriptional memory with increased passage number: • Passive replication dependent loss of somatic marks in majority of iPSCs • Selection of rare, pre existing, fully reprogrammed cells that has both growth and survival advantage. • The tendency of early passage iPSCs to differentiate prefentially into cell lineage of origin could be potentially exploited in clinical settings to produce certain somatic cells that have been difficult to obtain with ESCs so far. • Recapitulation of disease phenotypes with early passage patient specific iPSCs with epigenetic, transcriptional and functional immaturity may confound data obtained from them.

  34. Summary

  35. Thank U

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