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The Alchemy of Induced Pluripotent Stem Cells

The Alchemy of Induced Pluripotent Stem Cells. Uma Ladiwala UM-DAE Centre for Excellence in Basic Sciences Kalina Campus, Mumbai. Alchemists 300 years ago tried, unsuccessfully, to turn base LEAD into valuable GOLD Cellular Alchemy:

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The Alchemy of Induced Pluripotent Stem Cells

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  1. The Alchemy of Induced Pluripotent Stem Cells Uma Ladiwala UM-DAE Centre for Excellence in Basic Sciences Kalina Campus, Mumbai

  2. Alchemists 300 years ago tried, unsuccessfully, to turn base LEAD into valuable GOLD Cellular Alchemy: Normally, stem cells give rise to somatic cells of the adult organism Recent developments have resulted in reversing this process with the production of stem cells from adult somatic cells, eg. skin cells These stem cells have been termed “Induced Pluripotent Stem (iPS) Cells” ALCHEMY

  3. What is a Stem Cell?- Properties An unspecialized cell with a unique capacity for - indefinite or prolonged self-renewal and - ability to give rise to differentiated cells

  4. Self-renewal - ability to undergo numerous cycles of cell division while maintaining the undifferentiated or unspecialized state Clonality – ability of a single cell to form many similar cells Differentiation - process by which a less specialized cell becomes a more specialized one. Potency- the potential for differentiation to specialized cell types What is Self Renewal? Differentiation?

  5. Potency of stem cells Pluripotent – give rise to cells of all 3 germ layers (ectoderm, endoderm, mesoderm and germ cells) Multipotent – ability to differentiate into many, related cell types Progenitors – oligopotent – few cell types unipotent – one cell type but can self renew

  6. At the Molecular Level Differentiated Cell Stem Cell Pluripotency genes on Differentiation genes off Pluripotency genes off Differentiation genes on

  7. Where are stem cells found? Stem cells have been isolated from the embryo, fetus and adult Embryonic stem (ES) cells: derived from the inner cell mass of the blastocyst (4-5 day embryo) Adult stem cells : from adult tissues

  8. Stem cell types and origins

  9. Stem cells-types and terminology Can form all tissues including placenta Embryonic Can form any embryonic tissue but not placenta Adult

  10. A : Stem cell B : Progenitor cell C : Differentiated cell 1 : Symmetric division 2 : Asymmetric division 3 : Progenitor cell division 4 : Terminal differentiation Division of Stem Cells

  11. Timeline of Stem Cell Research 1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; their reports are largely ignored. 1978 - Haematopoietic stem cells in human cord blood. 1981 - Mouse embryonic stem cellsare derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell". 1996 - Cloning of Dolly the sheep by somatic cell nuclear transfer 1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells. 1998 - James Thomson and coworkers derive the first human embryonic stem cell line 2000s - Several reports of adult stem cell plasticity 2004-2005 - Korean researcher Hwang Woo-Suk human embryonic stem cell line from unfertilised human oocytes by SCNT. The lines were later shown to be fabricated. August 2006 – Mouse Induced pluripotent stem cells: the journal Cell publishes Takahashi and Yamanaka’s work.

  12. What’s so special about Stem Cells? They have the potential to replace cell tissue that has been damaged or destroyed. They can replicate themselves over and over for a very long time- nearly inexhaustible source Understanding how stem cells develop into healthy and diseased cells will assist the search for cures. Drugs and chemicals can be screened and tested on patients’ stem cells and their differentiated tissues

  13. Embryonic Stem (ES) cells Derived from inner cell mass of blastocyst Capacity for almost unlimited symmetrical divisions without differentiation Give rise to endoderm, ectoderm, mesoderm Clonogenic- derived from a single ES cell Capable of colonizing germline and forming egg and sperm cells

  14. Cultivation of ES cells

  15. Characterization of human and mouse ES cells Expression of cell surface markers –SSEA-3,SSEA-4 (hESC) SSEA-1 (mESC),TRA-1-60, TRA-1-81, alkaline phosphatase, GTCM-2 - Pluripotency transcription factors – Oct-4, Sox-2, Nanog, Rex1 - High Telomerase activity - Karyotype- normal (46 XX or XY) - In vitro pluripotency- embryoid body formation - Teratoma formation in immune-incompetent mice- tumour contains tissues from all 3 germ layers - Pluripotency in vivo – Chimera formation (mESC)

  16. Derivation and Characterization of human ES cells AP Oct4 SSEA4 SSEA3 TRA-1-60 TRA-1-81 Chen et al, 2007

  17. Differentiation of human ES cells In-Vitro In-Vivo EB Neuronal cells Intestine Cardiac Mesoderm AFP Germ Respire Sk muscle Ova Cartilage Neural tube Chen et al, 2007 http://www.isscr.org/video/beatingMyocytes.mpg

  18. Embryonic SC “Pluripotent” Stable. Can undergo many cell divisions. Easy to obtain but blastocyst is destroyed (Ethics) Possibility of immune rejection High potential for tumours Adult SC “Multipotent” Less Stable. Capacity for self-renewal is limited. No ethical concerns Difficult to isolate in adult tissue. Host rejection minimized or absent Less tumorigenic potential Embryonic or Adult Stem Cells for Cell ReplacementTherapy: Advantages and Disadvantages

  19. The Ideal Stem cell – the “Holy Grail” of Cell Replacement Therapy - Ability to differentiate into many cell types - Easily accessible - Individual-specific i.e. personalized or non-immunogenic - Vastly renewable - Demonstrably safe - Non-tumorigenic

  20. The Induced Pluripotent Stem (iPS) Cell : A Likely Candidate?

  21. Re-programming the nucleus Stem cell Differentiation is not an irreversible commitment Stem cell Differentiated cell Nuclear reprogramming - functional or molecular changes in cells undergoing fate changes

  22. Reprogramming by somatic cell nuclear transfer and cell fusion Dolly the Sheep

  23. Transcription factors for reprogramming Transcription factors are proteins that bind to DNA and regulate gene expression Oct3/4 and Sox2: transcription factors that function in maintaining pluripotency in both early embryos and ES cells. c-Myc and Klf4: transcription factors that modify chromatin structure so that Oct3/4 and Sox2 can bind to their target; proto-oncogenes

  24. The making of iPS cells Cell trapping strategy: selection of Fbx15-neomycin-resistant cells What is fbx15 ? - a transcription factor in ES cells and early embryo but not essential for maintainence of pluripotency Takahashi and Yamanaka, Cell, Aug 25, 2006

  25. 24 candidate genes for pluripotency factors: Ecat1, Dpp5(Esg1), Fbx015, Nanog, ERas, Dnmt3l, Ecat8, Gdf3, Sox15, Dppa4, Dppa2, Fthl17, Sall4, Oct4, Sox2, Rex1, Utf1, Tcl1, Dppa3, Klf4, b-cat, cMyc, Stat3, Grb2 Takahashi and Yamanaka, Cell, Aug 25, 2006

  26. Takahashi and Yamanaka, Cell, Aug 25, 2006

  27. Takahashi and Yamanaka, Cell, Aug 25, 2006

  28. Were these iPS cells identical to the ES cells? NO - The transcriptional profile was somewhere between fibroblasts and ES cells - No live chimeras produced So these iPS cells were somewhat similar but not identical to ES cells WHY? Because fbx15 was selected for. Fbx15 is a factor that is expressed in ES cells but is not essential for the maintainence of pluripotency

  29. Is there a way to improve this and get ES –like iPS cells? Okita K et al, Nature, 2007

  30. Nanog-selected iPS cells -Expressed all markers and characteristics of ES cells -Chimera formation when injected into blastocysts but 20% of the mice developed tumours

  31. Proposed explanation for the difference

  32. Reprogramming: 2-stage process Vit C ESC morphology All pluripotent markers No somatic markers LIF responsive Chimera forming Germline competent ESC morphology Some pluripotent markers Loss of somatic markers LIF unresponsive No chimeras Germline incompetent

  33. Mechanism of ES cell pluripotency Oct4, Sox2m and Nanog form an interconnected auto-regulatory network

  34. Proposed mechanism of iPS cell reprogramming Exogenous Oct4 and Sox2 reactivate endogenous Oct4, Sox2 and Nanog and the auto-regulatory loop then becomes self-sustaining. Exogenous factors are silenced by DNA methylation Scheper, Copray, 2009

  35. Induced pluripotency : the two-stage switch Stage 2 Stage 1 Activation of auto-regulatory loop Full reactivation of ES cell transcriptional network Completion of transgene silencing Downregulation of lineage genes Activation of specific ES genes Chromatin remodelling

  36. Mouse Embryonic fibroblasts Adult tail fibroblasts Hepatocytes Gastric epithelial cell Pancreatic cell Neural stem cell B lymphocyte Keratinocyte Human Skin fibroblast Keratinocyte Bone marrow stem cell Peripheral blood cell iPS Cells- Starting cells

  37. Efficiency of re-programming is poor Hochdelinger and Plath, 2009

  38. Derivation of human iPS cells In human cells efficiency of reprogramming ranges between 0.02% to 0.002%

  39. Potentials of iPS cells - Ability to differentiate into many cell types - Easily accessible - Individual-specific i.e. personalized or non-immunogenic - Vastly renewable - Useful for studying mechanisms of disease - Useful for drug, toxicity testing

  40. iPS cell reprogramming: Problems Use of viral vectors for induction Low efficiency of reprogramming Risk of tumour formation Efficient differentiation protocols required

  41. Further work towards “safer” and more efficient generation of iPS cells Reduced number of transcription factor used: No myc: Nakagawa and Yamanaka, Nat Biotechnol 2008, Wernig and Jaenisch, Cell Stem Cell 2009 No Sox2: by adding GSK-3 inhibitor, Zhou and Ding, Stem cell 2009, in neural stem cell, Kim and Scholer Nature 2008 No Klf4/myc, by addition of Valproic acid : Huangfu and Melton, Nat Biotech 2008 No Myc and Sox2, by addition of BIX01294 and PD0325901 (Zhou and Ding, Cell Stem Cell 2008). Klf4 only by adding Kenpaullone (Lyssiotis and Jaenisch, PNAS 2009)

  42. Specific pathways: TGF-β inhibitor replaces Sox2 and cMyc and induce Nanog (Maherali and Hochedlinger, Curr Biol 2009, Ichida and Eggan 2009 ) p53 inhibition augments iPS efficiency (Hong and Yamanaka, Nature 2009,Utikal and Hochedlinger Nature 2009, Marion and Blastco Nature 2009, Li and Serrano Nature 2009, Kawamura and Belmonte 2009) Hypoxia stimulates iPS generation – Yoshida and Yamanaka Cell Stem Cell 2009 Wnt signaling stimulates reprogramming efficiency (Marsonm, Jaenisch Cell Stem Cell 2008)

  43. Better vectors: Drug Inducible vectors (Wernig and Jaenisch, Nat Biotechnol 2008, Hockemeyer and Jaenisch, Cell Stem Cell 2008) Non-integrating vectors adenovirus in hepatocyte (Stadtfeld and Hochedlinger, Science 2008) Multi-cistronic vectors: single lentiviral cassette ( Carey and Jaenisch, PNAS 2009, Sommer and Mostoslavsky, Stem Cell 2009) Vector free (episomes, Yu and Thomson, Science 2009; direct transfection, Okita and Yamanaka Science 2008) Direct protein induction: poly arginine modification of recombinant protein (Zhou and Ding, Cell Stem Cell 2009),

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